Genetically modified rodents and rodent cells, and uses thereof

Abstract

Transgenic rodents and rodent cells that express components of the human immune system, and their uses in generating human T cell receptors (TCRs). A genetically modified rodent comprising a TCR alpha gene locus comprising unrearranged human TCR alpha variable gene segments and a human TCR alpha constant region, and a TCR beta gene locus comprising unrearranged human TCR beta variable gene segments and a human TCR beta constant region. Use of hTCR transgenic rodents to generate antigen-specific CD8+ T cell responses for the discovery of fully human TCR sequences with therapeutic potential.

Description

[0001] Genetically modified rodents and rodent cells, and uses thereof

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a rodent, and rodent cells, that are genetically engineered to express components of the human immune system. The invention also relates to methods for making and using said rodents to generate therapeutically useful products, and to products identified and obtained using the rodents.

[0004] BACKGROUND OF THE INVENTION

[0005] There remains a need for in vivo systems that mimic aspects of human immune system.

[0006] Publications have been made concerning genetically modified mice that encode chimaeric (humanmouse) versions of the T cell receptor (herein "TCR") as well as chimaeric versions of TCR co-receptors such as CD4 and CD8, as well as chimaeric MHC class I and MHC class II. These publications include, inter alia, EP2958937 and EP2958938B1. Such chimaeric TCR polypeptides require subsequent humanisation, to replace the rodent portions to generate human therapeutics, and the resulting TCR variable regions are therefore never provided in exactly the same way as they were generated in vivo in the mouse. The chimaeric loci are generated with mouse polypeptide portions to interact with mouse control sequences and / or other mouse polypeptide sequences.

[0007] T cells express variable T cell receptors (TCRs) complexed with CD3 polypeptides. The TCR is activated via binding to its cognate peptide-major histocompatibility complex (pMHC), a cell surface component which is found on most mammalian cells and which presents a sample of peptides from within the cell via the antigen processing and presentation pathway. MHC class I molecules on the surface of most mammalian cells present 8 to 11 mer peptides derived from the cell's cytosolic proteins, and are recognised by CD8+ T cells in which the TCR-CD3 complex and co-receptor CD8 bind to the pMHC. See Figure 8.

[0008] A repertoire of TCRs with diverse sequences are generated by somatic rearrangement of genomic DNA within a population of T cells in vivo, each T cell expressing a single (monoclonal) TCR containing random sequence elements. Positive and negative selection during T cell development permits survival of T cells expressing a TCR which recognise pMHC with a threshold affinity and deletes self-reactive cells that exhibit high affinity binding, thereby producing a population of T cells which do not recognise normal "self" pMHC but which may bind previously unencountered pMHC. When a TCR of a CD8+ cytotoxic T lymphocyte (CTL) recognises a peptide displayed on MHC class I, the binding of the TCR to the pMHC initiates intracellular signalling via the CD3 component of the TCR complex, activating the CTL to release cytolytic molecules and immune cell-activating cytokines. Cells expressing proteins with neoepitopes (e.g., infected cells producing viral protein, and cells expressing mutated oncogenes) can thus be eliminated by the cellular adaptive immune system to maintain health.

[0009] Structurally, the TCR comprises two transmembrane polypeptides, which may be an a pair or a y6 pair, associated with the CD3 complex. Each of the two paired TCR polypeptide chains has an N terminal variable domain comprising three hypervariable complementarity determining regions (CDRs), a constant domain, and a connecting region linked to a transmembrane domain and C terminal cytosolic tail. Figure 9. In a structure analogous to an antibody's antigen-binding fragment (Fab) binding domain, the extracellular region of the TCR comprises two paired variable domains and two paired constant domains, and the pMHC binding site of the TCR is formed by a set of 6 CDRs, comprising three CDRs from one variable domain and three CDRs from the second variable domain.

[0010] A repertoire of different TCR variable domain sequences is generated from an individual genome, with sequence diversity within the variable domains being created by combining gene segments that are each individually selected from a plurality of different germline gene segments within the genetic locus encoding each TCR polypeptide chain. The CDRs are especially diverse in sequence as these include non- templated junctions between rearranged gene segments. Further combinatorial diversity is created through pairing of the different variable domains (e.g. ap pairing) to form the binding site of the TCR.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention relates to:

[0013] A genetically modified rodent, comprising:

[0014] (i) an unrearranged T cell receptor (TCR) a variable gene locus comprising at least one human Va segment and at least one human Ja segment, wherein the TCRa variable gene is operably linked to a human TCRa constant gene sequence,

[0015] (ii) an unrearranged TCR variable gene locus comprising at least one human v segment, at least one human DP segment, and at least one human jp segment, wherein the unrearranged TCRP variable gene is operably linked to a human TCRP constant gene sequence wherein the unrearranged human T cell variable region gene segments are capable of rearranging to form genes that encode human T cell receptor variable domains, wherein the unrearranged TCRa variable gene locus is present at an endogenous rodent TCRa gene locus and the unrearranged TCRP variable gene locus is present at an endogenous rodent TCRP gene locus.

[0016] A genetically modified rodent cell, such as an ES cell, comprising:

[0017] (i) an unrearranged T cell receptor (TCR) a variable gene locus comprising at least one human Va segment and at least one human Ja segment, wherein the TCRa variable gene is operably linked to a human TCRa constant gene sequence,

[0018] (ii) an unrearranged TCRP variable gene locus comprising at least one human vp segment, at least one human DP segment, and at least one human jp segment, wherein the unrearranged TCRP variable gene is operably linked to a human TCRP constant gene sequence wherein the unrearranged human T cell variable region gene segments are capable of rearranging to form genes that encode human T cell receptor variable domains, wherein the unrearranged TCRa variable gene locus is present at an endogenous rodent TCRa gene locus and the unrearranged TCRP variable gene locus is present at an endogenous rodent TCRP gene locus, A genetically modified rodent T cell, comprising:

[0019] (i) an unrearranged T cell receptor (TCR) a variable gene locus comprising at least one human Va segment and at least one human Ja segment, wherein the TCRa variable gene is operably linked to a human TCRa constant gene sequence,

[0020] (ii) an unrearranged TCRP variable gene locus comprising at least one human vp segment, at least one human DP segment, and at least one human jp segment, wherein the unrearranged TCRP variable gene is operably linked to a human TCRP constant gene sequence wherein the unrearranged human T cell variable region gene segments are capable of rearranging to form genes that encode human T cell receptor variable domains, wherein the unrearranged TCRa variable gene locus is present at an endogenous rodent TCRa gene locus and the unrearranged TCRP variable gene locus is present at an endogenous rodent TCRP gene locus.

[0021] A genetically modified rodent antigen presenting cell comprising: either

[0022] (i) a nucleic acid sequence encoding a human MHC class I polypeptide present at an endogenous rodent MHC class I locus, or

[0023] (ii) a nucleic acid sequence encoding a chimaeric human-rodent MHC class I polypeptide present at an endogenous rodent MHC class I locus, suitably wherein the nucleic acid sequence encoding the chimaeric MHC class I has a rodent exon 4 and exons 1-3 and 5-7 are human; and

[0024] (iii) optionally, a nucleic acid sequence encoding a human P2 microglobulin polypeptide, which when present, is located at an endogenous rodent P2 microglobulin locus, wherein the cell expresses either the human MHC class I or the chimaeric MHC class I, and optionally human P2M polypeptides.

[0025] A method for making a genetically modified rodent that expresses a human T cell receptor comprising:

[0026] (i) inserting into an endogenous rodent TCRa variable gene locus an unrearranged human TCRa variable gene locus comprising at least one human Va segment and at least one human Ja segment operably linked to a human TCRa constant region;

[0027] (ii) inserting into an endogenous rodent TCRP variable gene locus an unrearranged human TCRP variable gene locus comprising at least one human vp segment, at least one human DP segment, and at least one human jp segment operably linked to a human TCRP constant region. A method for making a genetically modified fertile rodent that expresses a human T cell receptor, the method comprising deletion of the rodent TCR p from at least rodent v i to Dpi, including a deletion of one or more or all of serine proteases genes Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2 , wherein one or more or all of the deleted serine proteases genes Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2 are re-inserted into the rodent genome.

[0028] A method for making a fertile rodent comprising a genetic knock out of the endogenous T cell receptor beta chain polypeptide, the method comprising deletion of the rodent TCR p from at least rodent vpi to Dpi, including a deletion of one or more or all of serine proteases genes Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2 , wherein one or more or all of the deleted serine proteases genes Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2 are re-inserted into the rodent genome.

[0029] A method of producing a human T cell receptor to an antigen of interest comprising:

[0030] (i) optionally, making a rodent as disclosed herein,

[0031] (ii) immunizing a rodent as disclosed herein with the antigen of interest;

[0032] (iii) allowing the rodent to mount an immune response;

[0033] (iv) determining a nucleic acid sequence of the human TCR variable regions expressed by a T cell from the rodent reactive to the antigen of interest, optionally including a step of isolating the T cell and

[0034] (v) expressing the human T cell receptor, or human T cell receptor variable region in a cell; optionally further formulating the expressed human T cell receptor, or human T cell receptor variable region with a pharmaceutically acceptable excipient; or

[0035] (vi) inserting the nucleic acid encoding the human T cell receptor, or human T cell receptor variable region, into a cell, ex vivo or in vitro, such as into a human or animal cell, optionally wherein the cell containing the inserted nucleic acid is formulated for delivery to a human or animal, respectively; or

[0036] (vii) formulating a nucleic acid (e.g. RNA or DNA) encoding the human T cell receptor, or human T cell receptor variable region, with a suitably delivery vehicle, such as a lipid or liposome, for delivery to a patient in need thereof in vivo.

[0037] A method of treating an individual in need thereof, the method comprising

[0038] (i) delivery of a cell according to step (vi) of the above statement into the patient in need thereof, or

[0039] (ii) delivery of a nucleic acid (e.g. RNA e.g. mRNA or DNA) encoding the human T cell receptor, or human T cell receptor variable region formulated as in step (vii) above into a patient in need thereof.

[0040] Desirably, transgenic rodents according to the invention show functional expression of human TCRs and provide an in vivo T cell immune repertoire that is well suited for selection of candidate TCRs for use in medical applications, such as therapeutic use in humans. Functional expression of the transgenic loci in rodents may be demonstrated to confirm their effective performance, for example by assessing data on the usage of germline gene segments in TCRs expressed from the transgenic TCR alpha and beta loci, data on combinations of TCR gene segments (e.g. v and j gene segments), N-addition, and CDR3 lengths.

[0041] TCRs isolated from the transgenic rodents may be tested to determine their affinity for a target antigen (target peptide MHC complex), their specificity and selectivity of binding to the target, and their ability to transduce signalling upon binding to the target pMHC when expressed in T cells (as measured for example by a reporter gene assay in T cells expressing the TCR). For selection of candidate therapeutics in particular it is desirable to identify TCRs exhibiting high affinity for the target and / or high potency in signalling, and transgenic rodents of the invention represent an exceptionally rich source of such TCRs. Advantageously, transgenic rodents in accordance with the invention may allow the isolation of TCRs that exhibit higher affinity and selectivity for binding a human target pMHC complex, as compared with isolating TCRs from human individuals.

[0042] List of Figures

[0043] Figure 1 illustrates the mouse TRA locus, the human TRA locus, and the engineered human mouse TRA locus. Human DNA is from chromosome 14:21570693 to 14:22554820 (GRCh38) in the third panel. This represents an insertion of just under 1 Mbp of human DNA cloned from the 1.1 Mbp region illustrated in the middle panel, being inserted within the 1.95 Mbp region of mouse DNA illustrated in the top panel, leading to a replacement of 1.8 Mbp mouse DNA with ~ 1 Mbp human DNA. The inserted human DNA may contain 45 functional Va and 51 functional Ja gene segments (Figure la). Alternatively it may contain 44 functional Va gene segments and 50 functional Ja gene segments (Figure lb). In Figure lc, the illustration of the human TRD locus has been revised to show the human TRD gene segments (indicated by black triangles) at the depicted chromosomal region. In the human sequence there are 3 x TRD D genes, 4 x TRD J genes and 8 x TRD V genes (4 of the Vs are shared with the TRA repertoire).

[0044] Figure 2 illustrates the mouse TRB locus, the human TRB locus, and the engineered human mouse TRB locus in which the human DNA is inserted and the rodent DNA inverted, to preserve male fertility. Human DNA is from chromosome 7:142237898 to 7:142813740 (GRCh38) in the third panel. This represents an insertion of 576 kbp of human DNA, encompassing the 520 kbp illustrated in the middle panel. A 5.4 Mbp region of the mouse genomic DNA is inverted, introducing a separation of 4.8 Mbp between the first mouse vp gene segment (v i) and the first human v gene segment (vP2). The inserted human DNA may contain 48 functional vp gene segments and 13 functional jp gene segments (Figure 2a). Alternatively it may contain 43 functional vp gene segments and 13 functional jp gene segments (Figure 2b).

[0045] Figure 3a / b illustrates DNA encoding the human CD8a replacing DNA encoding mouse CD8a in the mouse genome and illustrates DNA encoding the human CD8P replacing mouse CD8P in in the mouse genome. A chimaeric CD8 polypeptide is generated. Figure 3b shows a process to make the genome of figure 3a in more detail. Figure 3a(ii) contains a correction of the genome co-ordinates shown in Figure 3a(i). Figure 3b(ii) contains a correction of the genome co-ordinates shown in Figure 3b(i). Figure 4 illustrates a hypothetical scheme by which DNA encoding the human CD8a could be used to replace DNA encoding mouse CD8a in the mouse genome and illustrates DNA encoding the human CD8P replacing mouse CD8P in in the mouse genome. A fully human CD8 polypeptide would be generated.

[0046] Figure 5a / b illustrates DNA encoding the human P2 microglobulin replacing DNA encoding mouse P2 microglobulin in the mouse genome. Figure 5b shows a process to make the genome of figure 5a in more detail. Figure 5a(ii) contains a correction of the genome co-ordinates shown in Figure 5a(i). Figure 5b(ii) contains a correction of the genome co-ordinates shown in Figure 5b(i).

[0047] Figure 6a / b illustrates the human HLA_A*02:01 MHC class I gene replacing mouse H2-D1 in the mouse genome. Figure 6b shows a process to make the genome of figure 6a in more detail.

[0048] Figure 7 illustrates the Rodent MHC class I region, showing in the second panel the deletion of H2- K and the knock in site for a human HLA-A to replace H2-D.

[0049] Figure 8 illustrates a representation of an immune synapse between a T cell (below) and a target cell (above). The TCR engages the pMHC complex across the synapse between adjacent cell membranes. The class I MHC is composed of an alpha chain comprising domains al, a2 and a3. N terminal domains al and a2 provide a binding site for peptide p. Beta 2 microglobulin (P2m) associates with the alpha chain. The TCR is composed of an a chain and a p chain. Each chain comprises an N terminal variable region, a constant region, a connecting region, a transmembrane region and a short cytosolic C-terminal tail. The TCR is complexed with CD3 polypeptides. Dimeric co-receptor CD8 of the T cell binds a3 domain of the MHC class I molecule. CD8 is a heterodimer of an a chain and a p chain, and is associated with intracellular signalling molecule Lek. The figure was created with BioRender.

[0050] Figure 9 illustrates a TCR in the cell membrane. The figure was created with BioRender.

[0051] Figure 10 TRBV and TRBJ transcripts detected from bulk sequencing of spleen of a representative humanized TRB mouse. Data shown are the number of unique molecular identifiers (UMI) rather than total reads. Only functional TRBV and TRBJ counts are shown. Nonfunctional genes are often, but not always, zero counts.

[0052] Figure 11 TRBV UMI from bulk sequencing of spleen RNA.

[0053] Figure 12 TRBJ UMI from bulk sequencing of spleen RNA.

[0054] Figure 13 TRAV and TRAJ transcripts detected from bulk sequencing of spleen of a representative humanized TRA mouse. Data shown are the number of unique molecular identifiers (UMI) rather than total reads. Only functional TRAV and TRAJ counts are shown. Nonfunctional genes are often, but not always, zero counts.

[0055] Figure 14 TRAV UMI from bulk sequencing of spleen RNA.

[0056] Figure 15 TRAJ UMI from bulk sequencing of spleen RNA. Figure 16 illustrates relative usage of TRBJ1-C1 and TRBJ2-C2 in transcripts from transgenic mice, compared with reported data for human. Transgenic mouse results are from bulk sequencing of spleen RNA from OpTiMus TRB transgenic mice.

[0057] Figure 17 compares TRBV expression from spleen RNA of human TRB transgenic mice with published data from human PBMC

[0058] Figure 18 compares TRBJ expression from spleen RNA of human TRB transgenic mice with published data from human PBMC

[0059] Figure 19 compares the distribution of T cell receptor CDR3 nucleotide lengths for (A) TRB in humans (Kitaura et al, 2016) (B) TRB in transgenic mice (C) TRA in humans (Kitaura et al, 2016) (D) TRA in transgenic mice.

[0060] Figure 20 compares TRAV expression from spleen RNA of human TRA transgenic mice with published data from human PBMC

[0061] Figure 21 compares TRAJ expression from spleen RNA of human TRA transgenic mice with published data from human PBMC

[0062] Figure 22 illustrates usage of (a) TRAV, (b) TRAJ, (c) TRBV and (d) TRBJ genes in hTCR transgenic mice. "I" indicates reported expression of the gene. "0" indicates reported absence of expression of the gene. indicates reported low expression (<0.01 %). "?" indicates absence of reported data.

[0063] Figure 23 shows detection of cells with surface expression of mouse CD3 (vertical axis) and human CD8b (horizontal axis) by FACS in OpTiMus TRA / TRB transgenic mice of various zygosities.

[0064] Figure 24 shows detection of splenocytes with surface expression of mCD3, mCD4, hCD8b, hTRBC- 1 and mCD19 in fully homozygous OpTiMus mouse 8. Data are representative of results from 3 mice (mouse 8, 9, 10).

[0065] Figure 25 shows splenocyte populations from naive OpTiMus mice (mouse 8, mouse 9 and mouse 10).

[0066] Figure 26 shows representative data from mice following peptide immunisation compared with data from a naive control mouse

[0067] Figure 27 shows immune responses in hTRA+ / + (homozygous) hTRB+ / - (heterozygous) mice following peptide immunisation

[0068] Figure 28 shows immune responses in hTRA+ / + hTRB+ / + fully homozygous mice following peptide immunisation

[0069] Figure 29 shows yield of 124 soluble TCR-Fc fusion polypeptides produced by recombinant expression in Expi293T cells. Each dot represents one TCR.

[0070] The Figures indicate certain preferred co-ordinates for the genome modification in mice, although these are not limiting on the invention. In particular, insertions of human DNA having the following co-ordinates are made (the inserted human DNA includes all of the numbered nucleotides and the mouse genome retains the numbered nucleotides in the genome).

[0071] Tra

[0072] Human DNA from position 14:21,570,693 to 14:22,554,820 (GRCh38)

[0073] Human DNA insertion located between mouse position 14:52,664,870 or position 14:52,664,818 (depending on mouse line) and 14:54,463,673 of (GRCm39)

[0074] Trb

[0075] Human DNA from position 7:142,237,898 to 7:142,813,740 (GRCh38)

[0076] Human DNA insertion located between mouse position 6:36,074,375 and 6:41,535,764 (GRCm39)

[0077] Inversion of mouse DNA from 6:40,868,163 to 6:41,515,110 located between mouse 6:36,070,006 and 6:36,074,375

[0078] MHC class I

[0079] Human DNA from position 6:29,942,554 to 6:29,945,455 (GRCh38)

[0080] Human DNA insertion located between mouse position 17:35,482,089 and 17:35,485,847 (GRCm39)

[0081] CD8A (Figure 3a(i))

[0082] Human DNA from position 2:86,789,408 to 2:86,790,726 (GRCh38)

[0083] Human DNA Insertion located between mouse position 6:71,350,536 and 6:71,351,769 (GRCm39)

[0084] CD8A (Figure 3a(ii))

[0085] Human DNA from position 2:86,790,825 to 2:86,789,408 (GRCh38)

[0086] Human DNA Insertion located between mouse position 6:71,350,536 and 6:71,351,769 (GRCm39)

[0087] CD8B (figure 3a(i))

[0088] Human DNA from position 2:86,861,751 to 2:86,853,004 (GRCh38)

[0089] Human DNA Insertion located between mouse position 6:71,299,840 and 6:71,306,867 (GRCm39)

[0090] CD8B (figure 3a(ii))

[0091] Human DNA from position 2:86,861,865 to 2:86,853,004 (GRCh38)

[0092] Human DNA Insertion located between mouse position 6:71,299,840 and 6:71,306,770 (GRCm39) B2M (figure 5a(i))

[0093] Human DNA from position 15:44,711,547 to 15:44,716,342 (GRCh38)

[0094] Human DNA Insertion located between mouse position 2:121,978,219 and 2:121,982,235 (GRCm39)

[0095] B2M (figure 5a(ii))

[0096] Human DNA from position 15:44,711,547 to 15:44,716,342 (GRCh38)

[0097] Human DNA Insertion located between mouse position 2:121,978,218 and 2:121,982,142 (GRCm39)

[0098] Figures are not to scale but use of / / or / / / indicates that regions of the chromosome have not been included in the figure.

[0099] In all Figures : Grey = Mouse Black = Human Enh = enhancer An unshaded white box at the 5' end of an exon (e.g. Fig 3a) is the 5' untranslated region, which is part of an exon but is not a part of the coding sequence.

[0100] Detailed description

[0101] The present invention relates to rodents and rodent cells that comprise human nucleic acid encoding one or more polypeptide components of the human T cell molecular apparatus inserted into the rodent genome at the endogenous mouse locus for that component. In particular, the rodents or cells comprise human nucleic acid encoding one or more, and preferably all of; a human TCRa polypeptide, a human TCRP polypeptide, a human MHC class I polypeptide, a human CD8a polypeptide, a human CD8P polypeptide and a human P2 microglobulin polypeptide. In another aspect the rodents or rodent cells also comprise human nucleic acid encoding a human TCR6 polypeptide. The TRCa polypeptide is encoded by the TRA locus and TCRP polypeptide encoded by the TRB locus, but these loci may also be referred to herein as the TCRa and TCR p locus.

[0102] Where there is a human TCR expressed on a cell (comprising the human TCRa polypeptide and a human TCRP polypeptide), that human TCR will interact with a peptide presented by an MHC molecule on an antigen presenting cell. The MHC class I al and a2 domains interact with the TCR. The MHC class I a3 domain binds the CD8 molecule that is a coreceptor for the TCR. These regions of the MHC class I and / or the CD8 coreceptor may be engineered for effective peptide presentation to the human TCR, as described herein.

[0103] A TCR as described and claimed herein comprising the human TCRa and / or human TCRP polypeptide, preferably a fully human TCR, may be combined in the genome with any of the other alleles described herein, which may be human, chimaeric or mouse. The examples of the other alleles described herein are only optional examples and other suitable alleles may be identified from the common general knowledge and / or earlier publications of these (human / chimaeric / mouse) alleles. Examples of other alleles that may be used with the TCR include those described in the following publications and patent applications, incorporated herein by reference:

[0104] Chimaeric MHC (HHD construct with human beta2m(B2m) fused to MHC comprising human alphal, human alpha2 and mouse alphas, TM and cytoplasmic regions). 1997 J. Exp. Med. 185(12):2043-2051, HLA-A2.1-restricted Education and Cytolytic Activity of CD8-T Lymphocytes from beta2-microglobulin (B2m) HLA-A2.1 Monochain Transgenic H-2Db B2m Double Knockout Mice. Steve Pascolo, Nathalie Bervas, Jan M. Ure, Austin G. Smith, Frangois A. Lemonnier, and Beatrice Perarnau.

[0105] MHC1 and / or CD8 alleles described in WO02 / 059263.

[0106] MHC1, B2m and / or CD8 alleles described in US2005066375 (W003006639A1).

[0107] MHC1 and / or B2m alleles of the ABabDII mouse, as described in:

[0108] (i) 2010 Nature Medicine 16(9):1029 Transgenic mice with a diverse human T cell antigen receptor repertoire, Liang-Ping Li, J Christoph Lampert, Xiaojing Chen, Catarina Leitao, Jelena Popovic, Werner Muller & Thomas Blankenstein;

[0109] (ii) 2015 Nature Biotechnology 33(4):402-407. Identification of human T-cell receptors with optimal affinity to cancer antigens using antigen-negative humanized mice Matthias Obenaus, Catarina Leitao, Matthias Leisegang, Xiaojing Chen, loannis Gavvovidis, Pierre van der Bruggen, Wolfgang Uckert, Dolores J Schendel & Thomas Blankenstein

[0110] MHC1 and CD8 alleles described in W02014 / 130671.

[0111] MHCI in 2016 Cancer Immunology Research 4(3):204, "Identification of T-cell Receptors Targeting KRAS- Mutated Human Tumors". Qiong J. Wang, Zhiya Yu, Kayla Griffith, Ken-ichi Hanada, Nicholas P. Restifo, and James C. Yang.

[0112] MHCI, B2m, CD8 alleles described in Moore et. AL, SCIENCE IMMUNOLOGY "Humanization of T cell- mediated immunity in mice", 6(66) 2021.

[0113] Suitable MHCI, B2m alleles as described in WO2021139799.

[0114] It is highly desirable to have usage of all or the vast majority of the inserted human V, D and J gene segments in the expressed human TCR chains in the rodent. In this way the full repertoire of human TCR molecules can potentially be expressed in the rodent. The rodents expressing human TCRs as described herein have been shown to use a significant number of the inserted human alpha and beta gene segments to make TCR chains.

[0115] In addition the rodents of the invention have been shown to express TCRP DJC2 from the inserted fully human Trb DNA, without the need to include murine intronic sequences.

[0116] The invention therefore relates to a rodent which expresses at least 35, at least 40, at least 41, at least 42, at least 43, at least 44 or all 45 of the inserted human variable Va gene segments in the TCR alpha chain repertoire of the rodent. The invention therefore relates to a rodent which expresses at least 35, at least 40, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50 or all 51 of the inserted human Ja gene segments in the TCR alpha chain repertoire of the rodent.

[0117] The invention therefore relates to a rodent which expresses at least 35, at least 40, at least 45, at least 46, at least 47 or all 48 of the inserted human variable vp gene segments in the TCR chain repertoire of the rodent.

[0118] The invention therefore relates to a rodent which expresses at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 or all 13 of the inserted human jp gene segments in the TCR p chain repertoire of the rodent.

[0119] The invention therefore relates to a rodent having a repertoire which includes TCR beta chains expressed from both the TRBDJC1 and the DJC2 gene cluster, suitably wherein there is no murine intronic sequence present in the inserted human DNA, such as no murine intronic sequence between the human D or J gene segments present in the genome.

[0120] One preferred aspect of the invention relates to a genetically modified rodent, comprising:

[0121] (i) an unrearranged T cell receptor (TCR) a variable gene locus comprising at least one human Va segment and at least one human Ja segment, wherein the TCRa variable gene is operably linked to a human TCRa constant gene sequence,

[0122] (ii) an unrearranged TCRP variable gene locus comprising at least one human vp segment, at least one human DP segment, and at least one human jp segment, wherein the unrearranged TCRP variable gene is operably linked to a human TCRP constant gene sequence wherein the unrearranged human T cell variable region gene segments are capable of rearranging to form genes that encode human T cell receptor variable domains, wherein the unrearranged TCRa variable gene locus is present at an endogenous rodent TCRa gene locus and the unrearranged TCRP variable gene locus is present at an endogenous rodent TCRP gene locus.

[0123] In a preferred aspect the rodent or rodent cell expresses a T cell receptor capable of interaction with a peptide presented on an MHC class I molecule in the rodent, suitably allowing the T cell to be activated and proliferate. In a preferred aspect the rodent or rodent cell expresses a CD8 protein which is capable of binding to an MHC class I expressed by the rodent. Preferably the region of the CD8 and MHC class I polypeptides that interact are from same species - e.g. both human or both rodent.

[0124] Other preferred aspects of the invention include:

[0125] A rodent comprising a genome encoding an MHC class I polypeptide in which the al and a2 domains are human.

[0126] A rodent wherein the MHC class I polypeptide comprises an a3 domain that binds CD8 expressed in the rodent.

[0127] A rodent wherein the a3 domain is human and the CD8 comprises a human MHC class I binding domain. A rodent wherein the CD8 comprises human CD8a.

[0128] A rodent wherein the CD8 comprises human-rodent chimaeric CD8a.

[0129] A rodent wherein the CD8 comprises human CD8p.

[0130] A rodent wherein the CD8 comprises human-rodent chimaeric CD8p.

[0131] A rodent comprising a genome in which the human or human-rodent chimaeric CD8 are present at an endogenous rodent CD8 locus.

[0132] A rodent wherein the MHC class I polypeptide comprises human transmembrane and cytoplasmic domains.

[0133] A rodent wherein the MHC class I a3 domain is rodent.

[0134] A rodent wherein the CD8 comprises endogenous rodent CD8.

[0135] A rodent wherein the DNA encoding the MHC class I polypeptide is integrated at the endogenous rodent MHC class I locus.

[0136] The invention also relates to methods for making such rodents and cells and the use of such rodents and cells in the generation of therapeutically useful molecules, such as human T cell receptors generated in the rodent disclosed herein, including obtaining the nucleic sequence of human TCRs from a rodent and expressing all, or the variable part of, or a binding portion of, the human TCR in a cell, such as a production cell line such as a CHO cell, or in a human or animal cell. Alternatively nucleic acid encoding such a human TCR, or the variable part of the TCR, or a binding portion of the TCR, may be the therapeutically useful molecule, appropriately formulated for delivery.

[0137] In certain aspects, the nucleic acid encoding the fully human polypeptide is provided in the rodent or rodent cell operably linked to one or more endogenous rodent control sequences, such as a rodent promoter, enhancer or other control sequences.

[0138] The rodent or rodent cell of the invention preferably does not express an endogenous rodent polypeptide from the rodent locus that has been modified to express the equivalent human polypeptide. In this way the inserted human sequence will form the majority or all of the expressed polypeptide within the rodent or from the cell. For example, in one aspect the inserted human TCRa polypeptide is expressed, but the endogenous rodent TCRa polypeptide is not expressed, from the endogenous TRA locus.

[0139] In preferred aspects of the above: preferably the rodent or cell disclosed herein does not express a functional endogenous TCRa polypeptide from the endogenous TCRa variable gene locus; preferably the rodent or cell disclosed herein does not express a functional endogenous TCRP polypeptide from the endogenous TCRP variable gene locus; preferably the rodent or cell disclosed herein does not express a functional endogenous rodent CD8 coreceptor from the endogenous CD8 locus; preferably the above rodent or cell disclosed herein does not express a functional endogenous rodent MHC class I polypeptide from the endogenous MHC class I loci; and / or preferably the above rodent or cell disclosed herein does not express a functional endogenous rodent P2 microglobulin from the endogenous rodent P2 microglobulin locus when the rodent or rodent cell comprises a nucleic acid sequence encoding a human P2 microglobulin.

[0140] Reference to "does not express", or 'no expression' of the endogenous polypeptide means that there is no significant / substantial expression of the rodent polypeptide, generally not more than 10% of the wild type expression of the endogenous rodent polypeptide. Preferably there is no expression of the rodent polypeptide from the modified locus and host polypeptide expression from the modified locus has been completely prevented. In this way the expression of the equivalent human polypeptide that has been inserted at the rodent locus may be maximised.

[0141] In one aspect the assessment of the level of expression is considered at the level of the modified locus, and therefore there may be expression from the other unmodified allele. However, it is preferred that there is no expression of the endogenous polypeptide which, for example, may be the result of a homozygous modification of both rodent loci. This applies for each modified locus independently. Therefore, in one aspect one or more or all loci referred to herein may be homozygous for the inserted human polypeptide and there is no expression from either allele at each locus. In one aspect one, two, three, four, five, six or all 7 loci disclosed herein (loci encoding a TCRa polypeptide, a TCRP polypeptide, a MHC class I polypeptide (including both a knockout or inactivating modification of both the rodent H2K locus and H2D locus), a human CD8a polypeptide, a human CD8P polypeptide and a human P2 microglobulin polypeptide) may be homozygous. In another aspect one or more or all loci disclosed herein may be heterozygotes, in which only one allele is modified. In one aspect one, two, three, four, five or all 6 loci disclosed herein may be heterozygous.

[0142] In certain aspects all or a part of the rodent nucleic acid locus (encoding the rodent polypeptide that is equivalent to the human polypeptide as described herein), is deleted from the rodent genome, to prevent expression of the host polypeptide, or the endogenous locus is inactivated, e.g. by inversion.

[0143] Preferably an inversion moves the rodent DNA, e.g. the rodent V gene segments, at least 0.5MB away from the native locus, such as at least 1 Mbp, e.g. at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, or at least 4.5 Mbp or more Mbp away from the native locus.

[0144] A preferred rodent expresses all of; a human TCRa polypeptide, TCRP polypeptide, MHC class I polypeptide, CD8a polypeptide, CD8 polypeptide and 2 microglobulin polypeptide, and does not express rodent TCRa polypeptide, TCR polypeptide, MHC class I polypeptide, CD8a polypeptide, CD8P polypeptide and P2 microglobulin polypeptide.

[0145] Other preferred rodents express: at least a human TCRa polypeptide and TCRP polypeptide, to form a functional TCR; a human TCRa polypeptide, TCRP polypeptide, MHC class I polypeptide, CD8a polypeptide, and CD8P polypeptide,

[0146] In one aspect the rodent or rodent cell genome comprises a fully human TCR after TCR gene segment rearrangement. Therefore, the fully human TCR can be expressed in the rodent or cell which does not need to be further humanised, which means that any potential impact of the subsequent humanisation process (e.g. the subsequent replacement of a mouse for human constant region) is eliminated. Such an approach minimises the impact of structural differences between a chimaeric TCR molecule and a fully human TCR, these differences resulting from the interface of the human Va / vp domain with the mouse Ca / cp domain in the chimaeric molecule.

[0147] The insertion of the human nucleic acid is targeted, and is made into the rodent genome at the endogenous rodent locus, which gives the possibility for naturally located endogenous control sequences to be used in combination with the inserted human DNA.

[0148] Human TCR 6 gene segments may also be inserted allowing for TCR6 chain expression, and a TCR6y to be expressed having at least a human 6 chain.

[0149] TCRa

[0150] Provided herein is a genetically modified rodent or rodent cell, such as an ES cell or T cell, comprising: an unrearranged T cell receptor (TCR)a variable gene locus comprising at least one human Va segment and at least one human Ja segment, wherein the TCRa variable gene is operably linked to a human TCRa constant gene sequence, wherein the unrearranged human T cell variable region gene segments are capable of rearranging to form genes that encode human T cell receptor variable domains, wherein the unrearranged TCRa variable gene locus is present at an endogenous rodent TCRa gene locus.

[0151] In one aspect of the invention, the T cell co-receptor polypeptide is expressed only on T cells of the rodent, e.g., it is not expressed on B cells of the rodent.

[0152] In one aspect of the invention, the T cell co-receptor polypeptide a and / or p is comprised in the germline of the rodent.

[0153] In one aspect, the rodent or rodent cell may be heterozygous for the nucleotide sequence encoding the human TCRa polypeptide.

[0154] In one aspect, the rodent or rodent cell may be homozygous for the nucleotide sequence encoding the human TCRa polypeptide.

[0155] In one aspect the inserted human TCRa nucleic acid comprises up to, or at least, 5, 10, 15, 20, 25, 30, 35, 40 or more TCR Va gene segments, such as up to 44 or 45 or at least 44 or 45 Va gene segments. In one aspect the inserted mouse TCRa nucleic acid comprises up to, or at least, 5, 10, 15, 20, 25, 30, 35, 40, 44 or 45 functional TCRVa gene segments. The inserted TCRa nucleic acid may also comprise nonfunctional TCR Va gene segments or pseudogenes.

[0156] In one aspect the inserted human TCRa nucleic acid comprises up to, or at least, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 51 TCR Ja gene segments. In one aspect the inserted mouse TCRa nucleic acid comprises up to, or at least, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 51 functional Ja gene segments. The inserted TCRa nucleic acid may also comprise non-functional TCR Ja gene segments or pseudogenes.

[0157] In one aspect the unrearranged TCRa variable gene locus comprises a complete repertoire of human Ja segments and a complete repertoire of human Va segments.

[0158] In one aspect the inserted human TCRa nucleic acid comprises all functional TCRVa gene segments and all functional Ja gene segments. The inserted human TCRa nucleic acid may comprise 44 functional TCRVa gene segments and 50 functional Ja gene segments. In one aspect the inserted human TCRa nucleic acid comprises 45 functional TCRVa gene segments and 51 functional Ja gene segments. In one aspect this rodent is a mouse.

[0159] TCRVa gene segments in the transgenic locus are optionally selected from the TRAV segments listed in Table 1. The TCRa variable gene locus may comprise some or all of the TRAV gene segments listed in Table 1, preferably including all functional TRAV gene segments. TRAV7 and / or TRAV18 may not present or, if present, may not be expressed. Optionally, the TCRa variable gene locus comprises any one or more, or all, of the following TRAV gene segments which are capable of rearranging with TRAJ gene segments to form genes that encode human T cell receptor variable domains: TRAV1-1, TRAV1-2, TRAV2, TRAV3, TRAV4, TRAV5, TRAV6, TRAV8-1, TRAV8-2, TRAV8-3, TRAV8-4, TRAV8-6, TRAV9-1, TRAV9-2, TRAV10, TRAV12-1, TRAV12-2, TRAV12-3, TRAV13-1, TRAV13-2, TRAV14 / DV4, TRAV16, TRAV17, TRAV19, TRAV20, TRAV21, TRAV22, TRAV23 / DV6, TRAV24, TRAV25, TRAV26-1, TRAV26-2, TRAV27, TRAV29 / DV5, TRAV30, TRAV34, TRAV36 / DV7, TRAV38-1, TRAV38-2 / DV8, TRAV39, TRAV40, TRAV41.

[0160] TCRJa gene segments in the transgenic locus are optionally selected from the TRAJ segments listed in Table 1. The inserted human TCRa nucleic acid may comprise some or all of the TRAJ gene segments listed in Table 1, preferably including all functional TRAJ gene segments. Optionally, the TCRa variable gene locus comprises any one or more, or all, of the following TRAJ gene segments which are capable of rearranging with TRAV gene segments to form genes that encode human T cell receptor variable domains: TRAJ3, TRAJ4, TRAJ5, TRAJ6, TRAJ7, TRAJ9, TRAJ10, TRAJ11, TRAJ12, TRAJ13, TRAJ14, TRAJ15, TRAJ16, TRAJ17, TRAJ18, TRAJ20, TRAJ21, TRAJ22, TRAJ23, TRAJ24, TRAJ26, TRAJ27, TRAJ28, TRAJ29, TRAJ30, TRAJ31, TRAJ32, TRAJ33, TRAJ34, TRAJ35, TRAJ36, TRAJ37, TRAJ38, TRAJ39, TRAJ40, TRAJ41, TRAJ42, TRAJ43, TRAJ44, TRAJ45, TRAJ46, TRAJ47, TRAJ48, TRAJ49, TRAJ50, TRAJ52, TRAJ53, TRAJ54, TRAJ56, TRAJ57.

[0161] In one aspect the inserted human TCRa nucleic acid forms a contiguous human insertion in the mouse genome.

[0162] In one aspect the deleted TCRa DNA includes the genomic DNA from Vai to TCRaC inclusive.

[0163] In one aspect the inserted human DNA comprises the DNA from human Vai to the TCRaC inclusive.

[0164] In one aspect the TCRa constant region is the human TCRa constant region.

[0165] In one aspect the inserted human TCRa gene locus is operably linked to the rodent enhancer which is located downstream (3') of the rodent TCRa constant region in the rodent genome, preferably being the rodent enhancer at its native position in the genome. In one aspect the unrearranged human T cell variable region TCRa V and J gene segments are operably linked with the endogenous rodent TCRa enhancer downstream of TCRa constant region.

[0166] In one aspect, the rodent or rodent cell comprises or retains endogenous rodent promoter and / or other regulatory elements that control expression of the human TCRa locus. In one aspect, the nucleic acid encoding the fully human polypeptide is provided in the rodent or rodent cell operably linked to one or more endogenous rodent control sequences, such as rodent promoter, enhancer or other control sequences. In one preferred aspect one or more promoters of the inserted human TCRa human V gene segments are human. In one aspect the expression of one or more human V or J gene segments of the TCRa locus is under the control of a human promoter. In one aspect all of the V promoters are human.

[0167] In one aspect there is a lack of a functional endogenous rodent TCRa variable locus, e.g. resulting from a deletion in the rodent genome, e.g. which may be (a) a deletion of all endogenous Va gene segments, (b) a deletion of all endogenous Ja gene segments, and (c) a combination thereof.

[0168] In one aspect the endogenous rodent TCRa locus is deleted from the rodent genome from the distal 3' V (Vai) to the rodent 3' constant region TCRaC, inclusive, leaving the rodent enhancer in the rodent genome downstream of the TCRaC operably, linked to the unrearranged human T cell receptor (TCR) a variable gene locus.

[0169] In one preferred aspect the rodent is a mouse and the enhancer 3' of the constant region is a mouse enhancer.

[0170] In one aspect the rodent or rodent cell retains an endogenous TCRa variable gene locus and / or an endogenous rodent TCRP variable gene locus, wherein any retained endogenous rodent TCRa variable gene locus is a non-functional locus and wherein any retained endogenous rodent TCRP variable gene locus is a non-functional locus.

[0171] In one preferred aspect there is no rodent TCRa constant region at the rodent TCRa gene locus.

[0172] In one aspect the locus shows proper spatial and temporal protein expression and supports T cell development and selection.

[0173] TCR 5

[0174] In one aspect a rodent or rodent cell, such as an ES cell or T cell, disclosed herein comprises a repertoire of unrearranged human TCR6 variable region segments at the rodent TCR a locus.

[0175] In one aspect the inserted human TCRa locus comprises an unrearranged T cell receptor (TCR) 6 variable gene locus comprising at least one human V6 segment, at least one J6 gene segment and at least one human Ja segment, wherein the TCR6 variable gene segments are operably linked to the human TCR6 constant gene sequence.

[0176] In one aspect the rodent or rodent cell comprises a complete repertoire of human V6, D6 and J6 gene segments in the rodent genome. In one aspect a rodent or rodent cell disclosed herein comprises a complete repertoire of human V6 segments, a complete repertoire of human D6 segments, and a complete repertoire of human J6 segments at the human TCRa variable locus.

[0177] In one aspect a rodent or rodent cell disclosed herein comprises a fully human V6 constant region, further wherein there is no rodent V6 constant region at the TCRa variable locus.

[0178] The DNA encoding TCR6 occurs naturally within the TCRa locus and therefore insertion of the whole human TCRA locus will naturally result in insertion of the human TCR6 gene segments, including the 6 constant region. In one aspect the inserted human DNA comprises the DNA from human Va-1 - TCRaC inclusive, such that all of the human TCR6 will be included in the rodent genome.

[0179] The human TCR6 nucleic acid described herein can be inserted into the genome even if the whole TCRa locus is not inserted. It is preferably inserted within the TCRa locus.

[0180] In one preferred aspect the rodent or rodent cell genome does not comprise the rodent TCR6 constant region.

[0181] In one aspect the human TCR6 locus is operably linked to a human enhancer and / or a human promoter (s). In one aspect all of the promoters of the TCR6 V gene segments are human.

[0182] In one aspect the enhancer 5' of the constant region (TRDC) is human.

[0183] The rodent or rodent cell preferably comprises an unrearranged human TCR6 locus at the unrearranged human TCRa locus.

[0184] In one aspect a modified rodent or rodent cell is provided as disclosed herein wherein the unrearranged TCRa variable gene locus replaces all or a portion of an endogenous rodent TCRa gene locus.

[0185] In one aspect the inserted human TCR6 nucleic acid forms a contiguous human insertion in the rodent genome.

[0186] In one aspect the locus shows proper spatial and temporal protein expression and supports T cell development and selection.

[0187] TCRP

[0188] Provided herein is a genetically modified rodent, or rodent cell, such as an ES cell or T cell, comprising an unrearranged TCRP variable gene locus comprising at least one human vp segment, at least one human DP segment, and at least one human jp segment, wherein the unrearranged TCRP variable gene is operably linked to a human TCRP constant gene sequence wherein the unrearranged human T cell variable region gene segments are capable of rearranging to form genes that encode human T cell receptor variable domains, wherein the unrearranged TCRP variable gene locus is present at an endogenous rodent TCRP gene locus.

[0189] In one aspect the human TCRP locus is operably linked to the rodent enhancer which is naturally located downstream (3') of the rodent TCRP constant region in a wild-type rodent. Suitably this enhancer is located at its native position in the rodent genome. In one aspect the human TCRP locus is operably linked to the human enhancer which is normally located downstream of the human TCRPC2 constant region in the human locus.

[0190] In one aspect one or more promoters of the inserted human TCRa human V gene segments are human. In one aspect all of the promoters are human.

[0191] In one aspect the rodent or rodent cell disclosed herein does not comprise a rodent vpi gene segment.

[0192] In one aspect the human insertion comprises human DNA from vpi - VP30 inclusive. In one aspect the inserted human comprises human DNA from human Prss58 - VP30 inclusive. In one aspect the inserted DNA comprises a genomic human fragment or fragments.

[0193] In one aspect the inserted human TCRP nucleic acid comprises up to, or at least, or exactly, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 43, 45, 46, 47, 48 or more gene segments. The inserted TCRP nucleic acid may also comprise non-functional TCR vp gene segments or pseudogenes.

[0194] In one aspect the inserted human TCRP nucleic acid comprises up to, or at least, or exactly, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 jp gene segments. The inserted TCRa nucleic acid may also comprise nonfunctional TCR Ja gene segments or pseudogenes.

[0195] In one aspect the inserted human TCRP nucleic acid comprises one or both DP gene segments.

[0196] In one aspect the inserted human TCRP nucleic acid comprises Dpi - jpi.1-1.6 and DP2 - jp 2.1-2.7 gene segments.

[0197] The inserted human TCRP nucleic acid may comprise 43 functional TCRVP gene segments and 13 functional jp gene segments. This rodent may be a mouse. In one aspect the rodent is a mouse and inserted human TCRP nucleic acid comprises 48 functional TCRVP gene segments and 13 functional jp gene segments.

[0198] TCRVP gene segments in the transgenic locus are optionally selected from the TRBV segments listed in Table 1. The TCRP variable gene locus may comprise some or all of the TRBV gene segments listed in Table 1, preferably including all functional TRBV gene segments. Optionally, the TCRa variable gene locus comprises any one or more, or all, of the following TRBV gene segments which are capable of rearranging with TRBJ gene segments to form genes that encode human T cell receptor variable domains: TRBV2, TRBV3-1, TRBV4-1, TRBV4-2, TRBV5-1, TRBV5-4, TRBV5-5, TRBV5-6, TRBV6-1, TRBV6-2, TRBV6-4, TRBV6-5, TRBV6-6, TRBV6-8, TRBV7-2, TRBV7-3, TRBV7-4, TRBV7-6, TRBV7-7, TRBV7-9, TRBV9, TRBV10-1, TRBV10-2, TRBV10-3, TRBV11-1, TRBV11-2, TRBV11-3, TRBV12-3, TRBV12-4, TRBV12-5, TRBV13, TRBV14, TRBV15, TRBV16, TRBV18, TRBV19, TRBV20-1, TRBV24-1, TRBV25-1, TRBV27, TRBV28, TRBV29-1, TRBV30.

[0199] TCRjp gene segments in the transgenic locus are optionally selected from the TRBJ segments listed in Table 1. The inserted human TCRP nucleic acid may comprise some or all of the TRBJ gene segments listed in Table 1, preferably including all functional TRBJ gene segments. Optionally, the TCRP variable gene locus comprises any one or more, or all, of the following TRBJ gene segments which are capable of rearranging with TRBV gene segments to form genes that encode human T cell receptor variable domains: TRBJ1-1, TRBJ1-2, TRBJ1-3, TRBJ1-4, TRBJ1-5, TRBJ1-6, TRBJ2-1, TRBJ2-2, TRBJ2-3, TRBJ2-4, TRBJ2-5, TRBJ2-6, TRBJ2-7.

[0200] In one aspect the inserted human TCRP nucleic acid comprises all of the functional TCRVP gene segments and all of the functional jp gene segments.

[0201] In one aspect the rodent is a mouse.

[0202] In one aspect the inserted human TCRP nucleic acid comprises human TRBC1 and human TCRBC2.

[0203] In one aspect the inserted human TCRP nucleic acid forms a contiguous human insertion in the mouse genome.

[0204] In one aspect, the rodent may be heterozygous for the nucleotide sequence encoding the human TCRP polypeptide.

[0205] In one aspect, the rodent may be homozygous for the nucleotide sequence encoding the human TCRP polypeptide.

[0206] In one aspect, the rodent or rodent cell comprises or retains endogenous rodent promoter, enhancer and / or other regulatory elements that control expression of the human TCRP locus. In one aspect, the nucleic acid encoding the fully human polypeptide is provided in the rodent or rodent cell operably linked to one or more endogenous rodent control sequences, such as rodent promoter, enhancer or other control sequences.

[0207] In one aspect the rodent is a mouse and the enhancer 3' of the constant region (TRBC) and 5' of Vb31 is a mouse enhancer.

[0208] In one aspect the inserted human locus shows proper spatial and temporal protein expression and supports T cell development and selection.

[0209] In one aspect there is a lack of a functional endogenous rodent TCRP variable locus, e.g. resulting from a deletion in the rodent genome, e.g. which may be (a) a deletion of all endogenous vp gene segments, (b) a deletion of all endogenous jp gene segments (c) a deletion of all endogenous J6 gene segments, (d) a deletion of the host rodent constant region(s) and (e) a combination thereof.

[0210] Preferably both the rodent TCRa and TCRP locus have a deletion as described herein, or one or both are inactivated by inversion as described herein.

[0211] In one aspect the rodent retains an endogenous TCRP variable gene locus wherein the retained endogenous rodent TCRP variable gene locus is a non-functional locus, for example, the endogenous locus is inverted.

[0212] Fertility

[0213] In a separate aspect the invention relates to the realisation that the TCR Beta locus contains genes that affect mouse fertility. We have observed (see Example 1) that mice in which the TCRbeta locus has been deleted to prevent host TCRbeta chain expression are less fertile than those comprising an inverted TCRbeta variable locus in which the mouse also makes no TCR beta chains but retains the DNA of the locus in the genome, some of which is in inverted orientation to the wild type.

[0214] In particular, homozygous mice with a duplicated TCRP locus (human TR VJ1D1C1J2J2C2 and mouse TrPvlJlDl) were able to breed better than mice having the deletion.

[0215] Analysis of the TCRbeta locus indicates that the fertility is likely to be caused by deletion of one or more or all of the serine proteases Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2 located in the deleted region of the TCR beta locus.

[0216] Individual knock out of PRSS58 does not cause fertility problem in mice (Park et. al, 2020 Biology of Reproduction, 103(2), 195-204), and therefore deletion of this gene is not thought to be the cause, or the sole cause, of the observed reduction in fertility.

[0217] Accordingly, further aspects of the invention relate to rodents containing a modification in the TCRP locus which knocks out expression of the TCRP chain, where the rodents e.g. mice retain in their genome one or more, or all, of the said serine protease genes from the rodent e.g. mouse TCRpv region of the locus. The serine protease gene or genes may be retained in their endogenous position in the locus (for example, where the modification is of a part of the TCRP locus which does not affect expression of the serine protease gene or genes), or may be moved as part of an inversion of the TCRpv region, or may be excised from the endogenous position (for example as part of a deletion of the TCRpv region, e.g., deletion of the TCRP locus) and inserted in the genome at an ectopic position.

[0218] In one aspect the host TCR beta constant region is not inverted.

[0219] Male rodents preferably retain fertility comparable to wild type, which may optionally be measured by litter size.

[0220] The serine protease gene or genes used to restore fertility as described herein may alternatively be obtained or obtainable from the DNA of the host rodent strain or from other rodents who comprise gene equivalents of these serine proteases.

[0221] The present invention therefore relates to a rodent or rodent cell (preferably a mouse or mouse cell e.g. ES cell) in which the host TCR beta locus has been inactivated such that the rodent does not make a native rodent TCRbeta chain, but retains wild type or substantially wild type levels of fertility. Such wild type or substantially wild type fertility may be achieved either by retaining the TCRbeta locus DNA in the genome of the rodent in a form that does not express the TCRbeta polypeptide (e.g. having some or all the variable region in an inverted genomic orientation with respect to any TCRbeta constant region), and thereby retain the serine protease genes, or by deletion of all or a part of the locus sufficient to prevent native rodent TCRbeta chain expression, and re-introducing into the rodent genome any one of more or all of the Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2 genes from the rodent TCRbeta locus that have been deleted, preferably all of these genes if the TRBeta locus has been deleted.

[0222] It will be apparent that where there has been a deletion or other inactivation of TCRbeta locus which results in a concomitant reduction in fertility, the replacement of each of the deleted or inactivated serine protease genes can be carried individually, or in limited gene combinations, to determine which gene or genes are needed to restore fertility. It may not be necessary to include all of the serine protease genes (Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2). Therefore skilled person can - by a practical assessment of fertility, e.g. by litter size - determine which of the serine protease genes (Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2) are required to restore fertility.

[0223] The host TRB locus may also be effectively inactivated in other ways that retain the expression of the fertility gene(s), such as by moving the variable portion of the locus further away from the constant region, e.g. by insertion of DNA, such as human variable TCR beta DNA, between the mouse TCR beta DNA and the constant region.

[0224] In one aspect the rodent TCRP gene locus is deleted from the genome, such that the Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2 genes which occur within the locus are deleted, and then one, or more, or all of these genes are replaced suitable to restore fertility in a male rodent, in combination with the insertion of DNA encoding at least one human vp segment, at least one human DP segment, and at least one human jp segment, as disclosed herein. The host TCR beta locus is inactivated and a human TCRbeta variable region can be expressed, preferably as a fully human chain in combination with a human constant region.

[0225] The invention also relates to a method for replacing a rodent TCRP locus with a human TCRP locus, the method comprising replacing the TCRP rodent locus with the human TCRP locus and then re-introducing into the rodent genome a rodent serine protease gene, such as one or more or all of Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2 genes, optionally being the same genes as the genes deleted from the rodent. In one aspect of the method the rodent locus that is deleted includes the vpi gene segment, and / or the region between vpi and VP2. In one aspect of the method the rodent DNA that is deleted includes the genomic region between VP29 and Dpi. The host TCR beta locus is inactivated.

[0226] The invention also relates to a genetically modified male rodent having substantially wild type fertility, wherein the rodent TCRP locus has been deleted from at least rodent vpi to Dpi, and the genome additionally comprises one or more or all of the deleted rodent serine proteases genes Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2, wherein the rodent genome is not a wild type rodent, or does not have a wild type genome at the TCRP locus.

[0227] The invention also relates to a rodent or rodent cell expressing a fully human TCRalpha and / or fully human TCRbeta chain as disclosed herein, wherein the rodent retains an endogenous TCRa variable gene locus and / or an endogenous rodent TCRP variable gene locus, and wherein any retained endogenous rodent TCRa variable gene locus is a non-functional locus and / or wherein any retained endogenous rodent TCRP variable gene locus is a non-functional locus, optionally wherein all or a part of the retained endogenous rodent TCRa and / or p variable gene locus is inverted with respect to its normal orientation in the rodent genome.

[0228] The invention also relates to a fertile rodent or rodent cell wherein the rodent TCRP locus from at least rodent vpi to Dpi is inverted within the rodent genome with respect to the natural orientation in the rodent genome, optionally the method further comprising the insertion into a rodent genome of nucleic acid, such as DNA, encoding all or a part of a TCRP from another species which is different from the rodent, such as inserting human DNA encoding all or a part of TCRp. The invention also relates to a genetically modified rodent or rodent cell wherein the rodent retains the endogenous rodent TCRP variable gene locus, and wherein the endogenous rodent TCRP variable gene locus is a non-functional locus as a result of inversion of all or a part of the locus within the genome.

[0229] Other aspects of the invention include:

[0230] Rodents e.g. mice and cells according to any disclosure herein in which the TRB VDJ locus is retained in the genome and not deleted.

[0231] Any method or rodent or rodent cell as disclosed herein wherein the cell does not express a host rodent TCRP chain.

[0232] A genetically modified male rodent having substantially wild type fertility, wherein all or a part of the variable VDJ region of the rodent TCRP locus is inverted within the rodent genome with respect to the natural orientation in the rodent genome.

[0233] A rodent cell, such as an ES cell capable of development into a male rodent having substantially wild type fertility, wherein all or a part of the variable VDJ region of the rodent genome TCRP locus is inverted within the rodent genome with respect to the natural orientation in the rodent genome.

[0234] Optionally the rodent genome, such as a male rodent genome, comprises any TCR alpha, TCR beta, MHC class I, CD8 alpha, CD8beta or beta 2 microglobulin allele or alleles as described herein.

[0235] The invention also relates to a method for making a fertile rodent, the method comprising the step of inverting the rodent TCRP in the rodent genome from at least rodent vpi to Dpi, optionally the method further comprising the insertion into a rodent genome of nucleic acid, such as DNA, encoding all or a part of a TCRP from another species which is different from the rodent, such as inserting human DNA encoding all or a part of TCRp.

[0236] The invention also relates to a method for making a fertile rodent, the method comprising, in either order

[0237] (i) deletion of the rodent TCRP genomic DNA from at least rodent vpi to Dpi, including a deletion of one or more or all of the serine proteases genes Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2, and

[0238] (ii) insertion of one or more or all of the serine proteases genes Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2 into the rodent genome.

[0239] In one aspect is provided a genetically modified rodent or rodent cell as disclosed herein wherein the rodent TCRP locus is deleted from at least rodent vpi to Dpi, and one or more or all of the deleted serine proteases genes Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2 are reinserted into the rodent genome, wherein the rodent is not a wild-type rodent.

[0240] In one aspect is provided a modified rodent or rodent cell disclosed herein wherein an unrearranged human TCRP variable gene locus replaces all or a portion of an endogenous rodent TCRP gene locus. Human intronic / intergenic DNA

[0241] In one aspect the inserted TCRP locus comprises fully human TCRP D and J gene segments including introns and intergenic sequences. In the present invention no rodent DNA was required for expression of the TCRP DJC2 gene cluster, unlike the issue observed in Moore et al [Moore et al., Sci. Immunol. 6, eabj4026 (2021) 17 December 2021], which notes, under the heading of "gene humanisation in mice" on page 8 of 11, that "An initial Trb humanization replaced coding, intergenic, and intronic regions with human sequences. These mice expressed a diverse TRBV repertoire, but only TRBDJCl and not DJC2 gene cluster utilization was detected. To address this, intergenic and intronic sequences were remurinized in this region (starting from steps 5 and 6 in fig. S4), resulting in robust usage of both DJC clusters (fig. S12B)".

[0242] An example of the variable TRB locus containing human introns is shown in Figure 2, in which human genomic TCRB variable region genomic TCRP DJC2 DNA has been inserted into the rodent genome, the inserted DNA comprising all of the human intronic sequences between D and J gene segments.

[0243] In one aspect not all of the human TCRP DJC2 D and J intronic sequences need to be present and some rodent D and J intronic sequences may be used in conjunction with human intronic sequences.

[0244] Example 2 confirms that the human intronic sequences located between TCRP DJC2 D and J gene segments, when inserted into the mouse genome, are sufficient for expression of the human V, D and J gene segments.

[0245] Therefore the present invention comprises a rodent or rodent cell having a TCRP locus as described herein comprising fully human TCRP DJC2 J introns and / or fully human TCRP DJC2 D introns, In one aspect the invention comprises a rodent with fully human TCRP DJC2 D and TCRP J gene segments, including fully human TCRP J introns and fully human TCRP D human introns. In one aspect no rodent TCRP DJC2 J introns and / or rodent TCRP DJC2 D introns are present in the genome.

[0246] The rodent of the invention is preferably also able to express a TCR receptor derived from a human gene segment located in the inserted human DJC2 gene cluster.

[0247] Therefore the present invention further comprises a rodent or rodent cell having a TCRP locus as described herein comprising fully human TCRP intergenic sequences between one or more or all of the D 1 and JI, JI and Cl, Cl and D2 and D2 and J2 gene segments. In one aspect no rodent TCRP intergenic segments are present in the genome between the DI and C2 gene segments, and preferably the insertion is a contiguous genomic human insert between human DI and human C2.

[0248] Therefore the present invention further comprises a rodent or rodent cell, optionally which is any as described herein, having a genome comprising DNA encoding a fully human TCR beta chain and comprising human V, D, J and C gene segments, wherein the rodent or rodent cell is capable of expression of said fully human TCR beta chain, and wherein the rodent genome comprises a human intergenic and / or a human intronic DNA sequence located between the human DI and human C2 gene segments, preferably being a human intergenic and / or intronic sequence that naturally occurs together with the human D and J gene segments, optionally wherein all of the human DNA sequence between the human DI and human C2 gene segments is genomic human DNA.Therefore, in one aspect, rodents e.g. mice, e.g. rodents and mice of the invention are preferred comprising one or more human TRB D and J introns at the mouse TRB DJC2 cluster, preferably comprising only human introns at this DJC2 cluster.

[0249] Therefore, in one aspect, rodents e.g. mice, e.g. rodents and mice of the invention are preferred that do not contain any murine TRB D and / or J introns at the mouse TRB DJC2 cluster.

[0250] Human CD8a and CD8P

[0251] The invention further relates to a genetically modified rodent, or rodent cell, such as an ES cell or T cell, comprising a nucleic acid sequence encoding a human CD8a polypeptide, wherein the nucleic acid sequence encoding a human CD8a polypeptide is present at an endogenous rodent CD8a co-receptor locus, and / or a genetically modified rodent, or rodent cell, such as an ES cell or T cell, comprising a nucleic acid sequence encoding a human CD8P polypeptide, wherein the nucleic acid sequence encoding a human CD8P polypeptide is present at an endogenous rodent CD8P co-receptor locus.

[0252] CD8 interacts with MHC class I molecules. CD8 may be a heterodimer of an a chain and a p chain or a homodimer of a or chains. The CD8 dimer on the T cell surface interacts with MHC class I of an adjacent cell (Figure 8). CD8 binding enhances the affinity of TCR binding to MHC class I . CD8 binding to MHC class I molecules is species-specific. Therefore the use of human CD8 is beneficial for studying T cell responses to antigen presented by human MHC class I.

[0253] Expression of CD8 is regulated by a variety of enhancer elements located throughout the CD8 locus. Regions of DNAse l-hypersensitivity which are associated with regulator binding have been identified at the CD8 locus. [Hosert et al. (1997) J. Immunol. 158:4270-81] . Enhancer elements have also been identified in the CD8 locus. [Kioussis et al. (2002) Nature Rev. 2:909-919 and Online Erratum; Ellmeier et al. (1998) Immunity 9:485-96],

[0254] In some aspects, the rodent or rodent cell comprises or retains endogenous rodent promoter, enhancer and / or regulatory elements that control expression of human CD8a and / or CD8p. In some aspects, the nucleic acid encoding the fully human CD8a or CD8P polypeptide is provided in the rodent or rodent cell operably linked to one or more endogenous rodent control sequences, such as rodent promoter, enhancer or other control sequences.

[0255] In one aspect the human CD8a and / or human CD8P are operably linked to a rodent promoter.

[0256] In one aspect, the rodents of the invention do not express CD8 protein on immune cells other than cells that normally express CD8, e.g., rodents do not express CD8 on B cells or CD4+ SP T cells.

[0257] The rodent or rodent cell e.g. a rodent T cell does not express a functional endogenous mouse CD8a and / or CD8P polypeptide from its endogenous CD8 locus.

[0258] In one aspect the rodent or rodent cell that expresses a functional CD8aP protein from a CD8 locus as described herein displays the human protein on a cell surface. In one aspect the rodent expresses the human CD8 protein on a cell surface in a cellular distribution that is the same or substantially the same as observed in a human. In one aspect, the human CD8 protein expressed in the rodent is capable of interacting with an MHC class I protein, such as a human MHC class I, expressed on the surface of a second cell. In one aspect the endogenous rodent CD8a and CD8P co-receptor locus comprises the inserted human DNA encoding CD8a and CD8P nucleic acid operably associated with at least one mouse enhancer, optionally with two or three mouse enhancers, and optionally also operably associated with one or more human enhancers, such that the CD8 locus (expressing both CD8a and CD8P) is under control of both human and rodent regulatory elements.

[0259] In one preferred aspect the rodent is a mouse and there are 4 mouse enhancers.

[0260] In one aspect, the rodent or rodent cell may be heterozygous for the nucleotide sequence encoding the human CD8a polypeptide. In one aspect, the rodent or rodent cell may be homozygous for the nucleotide sequence encoding the human CD8a polypeptide.

[0261] In one aspect, the rodent or rodent cell may be heterozygous for the nucleotide sequence encoding the human CD8P polypeptide. In one aspect, the rodent or rodent cell may be homozygous for the nucleotide sequence encoding the human CD8P polypeptide.

[0262] In one aspect the rodent or rodent cell comprises an insertion of (from exon 1-4) of the human CD8 a and from (exon 1-3) of human CD8p.

[0263] In one aspect the inserted human locus shows proper spatial and temporal protein expression and supports T cell development and selection.

[0264] Chimaeric CD8a and CD8

[0265] In an alternative aspect, the invention further relates to a genetically modified rodent, or rodent cell, such as an ES cell or T cell, comprising a nucleic acid sequence encoding a chimaeric rodent - human CD8a polypeptide (a chimaeric CD8a polypeptide), wherein the nucleic acid sequence encoding the chimaeric CD8a polypeptide is present at an endogenous rodent CD8a co-receptor locus, and / or a genetically modified rodent, or rodent cell, such as an ES cell or T cell, comprising a nucleic acid sequence encoding a chimaeric rodent- human CD8P polypeptide (a chimaeric CD8 p polypeptide), wherein the nucleic acid sequence encoding the chimaeric CD8P polypeptide is present at an endogenous rodent CD8P co-receptor locus

[0266] The chimaeric CD8a polypeptide is suitably encoded by genomic DNA which comprises a part of, or all of human exon 1, all of human exon2, all of human exon 3, and a part of human Exon 4, in one aspect the human DNA from human exon 4 being fused to a part of mouse exon 3. Preferably the rodent genome additionally comprises the human genomic DNA between Exons 1-2, Exons 2-3 and Exons 3-4.

[0267] In one aspect the fusion between human exon 4 and mouse exon 3 is made such that the extracellular part of the expressed polypeptide is human and the intracellular part of the polypeptide is mouse. The fusion suitably occurs in the connecting region just N terminal to the transmembrane domain.

[0268] In one aspect the fusion is made at a point at which the amino acid sequence (preferably the amino acid and DNA sequence) is identical between mouse and human so as to avoid introducing structural changes.

[0269] The CD8A DNA fusion suitably occurs human to mouse in the following region of the respective exons: Human Exon 4, bases 18 to 32 Mouse Exon 3, bases 18 to 32 The amino acid sequence for both species in this region is DFACD (i.e. Aspartic Acid, Phenylalanine, Alanine, Cysteine, Aspartic Acid).

[0270] The chimaeric CD8P polypeptide is suitably encoded by genomic DNA which comprises a part of or all of human exon 1, all of human exon2 and a part of human Exon 3, in one aspect the human DNA from exon 3 being fused to a part of mouse exon 3, and preferably the rodent genome additionally comprises the human genomic DNA between Exons 1-2 and Exons 2-3.

[0271] In one aspect the fusion between human mouse exons is made such that the extracellular part of the expressed CD8beta polypeptide is human and the intracellular part of the polypeptide is mouse.

[0272] In one aspect the human coding sequence but not the 5' untranslated region is comprised in the genome.

[0273] Figure 3 and 4 illustrates examples of suitable chimaeric CD8 polypeptides.

[0274] The chimaeric human-rodent CD8 protein is able to bind to the human MHC class I expressed by the host cells - for example a fully human MHC class I, to allow T cells expressing the chimaeric CD8 to interact with antigen presenting cells having MHC class I, thereby allowing presentation of antigen to the T cell and T cell activation. The ability of the chimaeric human-rodent polypeptide to bind with a human MHC class I can be achieved by retaining the regions of the human CD8 protein that are expressed on the T cell surface, and these sites of interaction with the human MHC class I are well known e.g. see Front. Immunol., 22 July 201 3Sec.TCellBiology, Volume 4 - 2013. T cell activation assays are also well known and can be used to determine T cell activation, and therefore confirm suitable chimaeric structures.

[0275] Expression of CD8 is regulated by a variety of enhancer elements located throughout the CD8 locus. Regions of DNAse l-hypersensitivity which are associated with regulator binding have been identified at the CD8 locus. [Hosert et al. (1997) J. Immunol. 158:4270-81], Enhancer elements have also been identified in the CD8 locus. [Kioussis et al. (2002) Nature Rev. 2:909-919 and Online Erratum; Ellmeier et al. (1998) Immunity 9:485-96],

[0276] In some aspects, the rodent or rodent cell comprises or retains endogenous rodent promoter, enhancer and / or regulatory elements that control expression of the chimaeric CD8a and / or CD8p. In some aspects, the nucleic acid encoding the chimaeric CD8a or CD8P polypeptide is provided in the rodent or rodent cell operably linked to one or more endogenous rodent control sequences, such as rodent promoter, enhancer or other control sequences.

[0277] In one aspect the chimaeric CD8a and / or chimaeric CD8P are operably linked to a rodent promoter.

[0278] In one aspect the chimaeric CD8a and / or chimaeric CD8P are operably linked to a human promoter.

[0279] In one aspect, the rodents of the invention do not express CD8 protein on immune cells other than cells that normally express CD8, e.g., rodents do not express CD8 on B cells or CD4+ SP T cells.

[0280] The rodent or rodent cell e.g. a rodent T cell does not express a functional endogenous mouse CD8a and / or CD8P polypeptide from its endogenous CD8 locus.

[0281] In one aspect the rodent or rodent cell that expresses a functional CD8aP protein from a CD8 locus as described herein displays the chimaeric protein on a cell surface. In one aspect the rodent expresses the chimaeric CD8 protein on a cell surface in a cellular distribution that is the same or substantially the same as observed in a human. In one aspect, the chimaeric CD8 protein expressed in the rodent is capable of interacting with an MHC class I protein, such as a human MHC class I, expressed on the surface of a second cell.

[0282] In one aspect the endogenous rodent CD8a and CD8P co-receptor locus comprises the inserted human DNA encoding CD8a and CD8P nucleic acid operably associated with at least one mouse enhancer, optionally with two or three mouse enhancers, and optionally also operably associated with one or more human enhancers, such that the CD8 locus (expressing both CD8a and CD8P) is under control of both human and rodent regulatory elements.

[0283] In one preferred aspect the rodent is a mouse and there are 4 mouse enhancers.

[0284] In one aspect, the rodent or rodent cell may be heterozygous for the nucleotide sequence encoding the chimaeric CD8a polypeptide. In one aspect, the rodent or rodent cell may be homozygous for the nucleotide sequence encoding the chimaeric CD8a polypeptide.

[0285] In one aspect, the rodent or rodent cell may be heterozygous for the nucleotide sequence encoding the chimaeric CD8P polypeptide. In one aspect, the rodent or rodent cell may be homozygous for the nucleotide sequence encoding the chimaeric CD8P polypeptide.

[0286] In one aspect the locus shows proper spatial and temporal protein expression and supports T cell development and selection.

[0287] EP2958938B1, derived from W02014130671, discloses examples of chimaeric MHC class I molecules and chimaeric CD8 polypeptides that are suitable for use in the present invention.

[0288] Rodent CD8a and CD80

[0289] In another aspect of the invention the host rodent CD8a and CD8P genes can be used. Preferably these are under the normal regulatory control of the host. In this aspect the rodent is used together in the genome with a chimaeric MHC class I, able to interact with the rodent CD8 protein via rodent amino acid portions regions present in the MHC class I polypeptide. The chimaeric MHC class I polypeptide suitably has a rodent alpha 3 domain, and the invention also relates to a nucleic acid encoding this chimaeric polypeptide.

[0290] Alternatively, host rodent CD8 is combined with MHC class I having a human alpha 3 domain. The rodent may comprise endogenous CD8 alpha and / or beta loci, each optionally homozygous, and an MHC class I locus encoding a human MHC class I polypeptide as described herein. Species mismatch between the (host) CD8 and the (human) MHC class I may reduce their interaction, compared with binding of species matched polypeptides (e.g., both human, or both mouse). Such rodents may exhibit weaker immune responses, generating fewer antigen-specific T cells to the target antigen, compared with rodents in which there is species match between CD8 and MHC class I a3 domain (e.g., both human, or both mouse). Nevertheless, antigen specific T cells obtained from such immunisations may bind the target antigen with high affinity. Reduced affinity of binding between the co-receptor and MHC may have an overall effect of selecting for higher affinity TCRs within the rodent, as a higher affinity TCR:MHC interaction would be required in order to meet the threshold for antigen-dependent TCR signalling within the T cell. This could be advantageous in the context of using the rodent to obtain T cell receptors with high affinity for certain antigens, for example to produce soluble TCR molecules for use as therapeutics, which act by binding pMHC in the absence of co-receptor engagement.

[0291] MHC class I

[0292] The invention further relates to a genetically modified rodent or rodent cell, such as an ES cell or antigen presenting cell, comprising a nucleic acid sequence encoding a human MHC class I polypeptide, and the nucleotide sequence encoding a human MHC class I polypeptide is present at an endogenous rodent MHC class I locus.

[0293] Such a rodent allows for a human CD8 protein expressed on the surface of a T cell of the rodent disclosed herein to interact with a human MHC class I expressed on a surface of a second cell, e.g., an antigen presenting cell in the rodent.

[0294] The human MHC I polypeptide may be derived from a human HLA class I protein selected from the group consisting of HLA-A, HLA-B, and HLA-C, e.g., HLA-A2, HLA-B27, HLA-B7, HLA-Cw6, or any other HLA class I molecule present in a human population.

[0295] The rodent genome can comprise more than one nucleic acid encoding a human MHC class I. For example, DNA encoding a human MHC class I can be inserted into both the rodent (e.g. mouse) H2D and H2K loci. The human MHC can be different at each locus. In one aspect there may be two or three or four different human MHC class I molecules present in the rodent genome, when considering the possibility of heterozygosity at each of these loci.

[0296] In some strains of rodent there are 3 rodent MHC class I loci and therefore there can be 1,2, 3, 4, 5 or even 6 different MHC class I molecules in the rodent genome, which can be human or humanised molecules and can be HLA-A, HLA-B and HLA-C .

[0297] In some aspects, the rodent or rodent cell comprises or retains endogenous rodent promoter, enhancer, and / or regulatory elements that control expression of human MHC class I.

[0298] In some aspects, the nucleic acid encoding the fully human MHC class I polypeptide is provided in the rodent or rodent cell operably linked to one or more endogenous rodent control sequences, such as rodent promoter, enhancer or other control sequences. In one aspect the rodent is a mouse and the human MHC class I polypeptide is operably linked to a mouse promoter and mouse enhancer located 5' of the fully human MHC class I polypeptide.

[0299] In one aspect, the rodent may be heterozygous for the nucleotide sequence encoding the human MHC class I polypeptide. In one aspect, the rodent may be homozygous for the nucleotide sequence encoding the human MHC class I polypeptide.

[0300] In one aspect the inserted human DNA, such as HLA-A02 DNA, is inserted at the endogenous H2-D locus. All or part of the endogenous H2-D locus may also be deleted or otherwise inactivated. In one aspect the rodent H2-K1 gene is also deleted or inactivated. In one aspect the inserted human DNA, such as HLA-A02 DNA, is inserted at the endogenous H2-K locus. All or part of the endogenous H2-K locus may also be deleted or otherwise inactivated. In one aspect the rodent H2-D gene is also deleted or inactivated.

[0301] Preferably both the rodent H2-D and H2-K genes are deleted or otherwise inactivated, such that mouse class I expression is eliminated, and only the desired human or humanised MHC class 1 molecule(s) are expressed in the rodent.

[0302] Preferably all rodent genes encoding MHC class I are deleted or otherwise inactivated, such that rodent class I expression is eliminated.

[0303] In one aspect the rodent comprises an insertion of nucleic acid encoding the complete coding sequence of human HLA-A02. In one aspect the inserted human DNA comprises human DNA comprising or consisting of nucleotides 6:29,942,554 - 29,945,450 (GRCh38).

[0304] In one aspect the human HLA-A02 is inserted at the endogenous H2-D locus.

[0305] In one aspect the rodent H2-K1 gene is deleted or inactivated.

[0306] In one aspect more that 1 rodent HLA loci may be inactivated. For example, the rodent may comprise an insertion of human DNA as described herein at one rodent MHC class I locus with inactivation of the endogenous expression at that locus, and may then also include inactivation (e.g. a knockout) of a second rodent MHC class I locus without replacement by a human MHC class I at a second locus. For example, the rodent H2-D locus may be replaced with nucleic acid encoding a human HLA class I gene and the rodent H2-K gene may be deleted without any human insertion at the H2-K locus.

[0307] In one aspect the inserted human locus shows proper spatial and temporal protein expression.

[0308] Chimaeric MHC class I

[0309] The invention further relates to a genetically modified rodent or rodent cell, such as an ES cell or antigen presenting cell, comprising a nucleic acid sequence encoding a chimaeric (rodent - human) MHC class I polypeptide, and the nucleotide sequence encoding a chimaeric MHC class I polypeptide is present at an endogenous rodent MHC class I locus.

[0310] Figure 6 illustrates one example of a suitable chimaeric MHC class 1 polypeptide.

[0311] The chimaeric MHC class I protein is able to bind to the rodent CD8 expressed by the host cells to allow T cells expressing the rodent CD8 to interact with antigen presenting cells having the chimaeric MHC class I, thereby allowing presentation of antigen to the T cell and T cell activation. The ability of the rodent CD8 polypeptide to bind with a chimaeric rodent-human MHC class I can be achieved by retaining a rodent region of the rodent MHC class I protein that interacts with the CD8, and these sites of interaction of rodent CD8 with rodent MHC class I are well known e.g. see Front. Immunol., 22 July 201 3Sec.TCellBiology, Volume 4 - 2013. T cell activation assays are also well known and can be used to determine T cell activation, and therefore confirm suitable chimaeric structures. EP2958938B1, derived from W02014130671, discloses examples of chimaeric MHC class I molecules and chimaeric CD8 polypeptides that are suitable for use in the present invention.

[0312] The MHC class I locus may therefore express a chimaeric (human-rodent) protein to allow the MHC class I to interact both with a human TCR but also to retain binding with the rodent CD8.

[0313] In particular where the CD8 is the endogenous rodent CD8, the MHC class I is preferably human except for the presence of a rodent alpha 3 domain, which is the site of interaction with CD8. Preferably the MHC I has human alpha 1 and human alpha 2 domains - these interact with the human TCR domains, so the MHC:TCR interface is fully human.

[0314] Therefore in one aspect the rodent comprises an insertion of nucleic acid encoding the complete coding sequence of human HLA-A02-01 Exons 1-3 and 5-8, and wherein exon 4 is the native rodent exon 4.

[0315] In one aspect the inserted human DNA comprises human DNA comprising or consisting of nucleotides 6:29,942,554 - 29,945,450 (GRCh38) - with the exception of human exon 4, which is instead provided as rodent exon 4 in the genome.

[0316] The whole chimaeric MHC class I may be inserted into the genome, or the rodent exon 4 existing in the genome may be retained, and the genome modified either side to introduce the desired human nucleic acid.

[0317] In one aspect the regions between human exons 1-2, 2-3, 3-4, 4-5, 5-6, 7-7 and 7-8 are all human.

[0318] Such a rodent allows for a rodent CD8 protein expressed on the surface of a T cell of the rodent disclosed herein to interact with the chimaeric (rodent - human) MHC class I expressed on a surface of a second cell, e.g., an antigen presenting cell in the rodent.

[0319] The human MHC I polypeptide may be derived from a human HLA class I protein selected from the group consisting of HLA-A, HLA-B, and HLA-C, e.g., HLA-A2, HLA-B27, HLA-B7, HLA-Cw6, or any other HLA class I molecule present in a human population.

[0320] The rodent genome can comprise more than one nucleic acid encoding a chimaeric MHC class I. For example, DNA encoding a chimaeric MHC class I can be inserted into the rodent (e.g. mouse) H2D and H2K loci. In one aspect there may be two or three or four different human MHC class I molecules present in the rodent genome, when considering the possibility of heterozygosity at each of these loci.

[0321] In some strains of rodent there are 3 rodent MHC class I loci and therefore there can be 1,2, 3, 4, 5 or even 6 different MHC class I molecules in the rodent genome, which can be chimaeric molecules and can be HLA-A, HLA-B and HLA-C .In some aspects, the rodent or rodent cell comprises or retains endogenous rodent promoter, enhancer, and / or regulatory elements that control expression of human MHC class I.

[0322] In some aspects, the nucleic acid encoding the fully human MHC class I polypeptide is provided in the rodent or rodent cell operably linked to one or more endogenous rodent control sequences, such as rodent promoter, enhancer or other control sequences. In one aspect the rodent is a mouse and the human MHC class I polypeptide is operably linked to a mouse promoter and mouse enhancer located 5' of the fully human MHC class I polypeptide.

[0323] In one aspect, the rodent may be heterozygous for the nucleotide sequence encoding the human MHC class I polypeptide. In one aspect, the rodent may be homozygous for the nucleotide sequence encoding the human MHC class I polypeptide.

[0324] In one aspect the inserted human DNA, such as HLA-A02 DNA, is inserted at the endogenous H2-D locus. All or part of the endogenous H2-D locus may also be deleted or otherwise inactivated. In one aspect the rodent H2-K1 gene is also deleted or inactivated.

[0325] In one aspect the inserted human DNA, such as HLA-A02 DNA, is inserted at the endogenous H2-K locus. All or part of the endogenous H2-K locus may also be deleted or otherwise inactivated. In one aspect the rodent H2-D gene is also deleted or inactivated.

[0326] Preferably both the rodent H2-D and H2-K genes are deleted or otherwise inactivated, such that mouse class I expression is eliminated, and only the desired human or humanised MHC class 1 is expressed in the rodent.

[0327] Preferably all rodent genes encoding MHC class I are deleted or otherwise inactivated, such that rodent class I expression is eliminated.

[0328] In one aspect more than 1 rodent HLA loci may be inactivated. For example, the rodent may comprise an insertion of human DNA as described herein at one rodent MHC class I locus with inactivation of the endogenous expression at that locus, and may then also include inactivation (e.g. a knockout) of a second rodent MHC class I locus without replacement by a human MHC class I at a second locus. For example, the rodent H2-D locus may be replaced with nucleic acid encoding a human HLA class I gene and the rodent H2-K gene may be deleted without any human insertion at the H2-K locus.

[0329] In one aspect the inserted human locus shows proper spatial and temporal protein expression.

[0330] P2M

[0331] The invention relates to a genetically modified rodent or rodent cell, such as an ES cell or antigen presenting cell comprising a nucleic acid sequence encoding a human P2 microglobulin polypeptide, which is located at an endogenous rodent P2 microglobulin locus.

[0332] In one aspect all or a part of the nucleic acid encoding the endogenous rodent P2 microglobulin is deleted.

[0333] In one aspect the rodent or rodent cell comprises an insertion of the ATG of exon 1 to the stop codon of exon 3. In one aspect the inserted human DNA comprises nucleotides 15:44,711,547 - 44,716,342 (GRCh38). In one aspect the insertion is into a mouse genome, deleting 2:121,978,219 - 121,982,140 (GRCm39 C57BL / 6J). In another aspect the insertion is into a mouse genome, deleting the region between 2:121,978,218 - 121,982,142 (GRCm39 C57BL / 6J), excluding these endpoints (i.e., the deleted region starts and ends 1 bp inside these flanking bases).

[0334] In some aspects, the rodent or rodent cell comprises or retains endogenous rodent promoter and / or regulatory elements that control expression of human P2M. In some aspects, the nucleic acid encoding the fully human P2M is provided in the rodent or rodent cell operably linked to one or more endogenous rodent control sequences, such as rodent promoter, enhancer or other control sequences. In one aspect the rodent upstream enhancer and promoter are operably linked to the nucleic acid encoding the P2M at the endogenous locus. Suitably the enhancer and promoter are the promoter and enhancer that are naturally present in the rodent or rodent cell, at the naturally occurring position in the genome, and have not been removed or replaced.

[0335] Preferably the rodent is a mouse and the enhancer is a mouse enhancer located 5' of the gene and promoter and shares cis and trans acting factors with MHC class I, with which expression is co-ordinated.

[0336] In one aspect, the rodent or rodent cell may be heterozygous for the nucleotide sequence encoding the human P2M polypeptide.

[0337] In one aspect, the rodent may be homozygous for the nucleotide sequence encoding the human P2M polypeptide.

[0338] In one aspect the inserted human locus shows proper spatial and temporal protein expression.

[0339] Combinations of human loci

[0340] Disclosed herein are rodents and cells comprising nucleic acid encoding a fully human polypeptide for one or more or all of the TCRa, TCRP, MHC class I, CD8a, CD8P and P2M loci which are inserted at the endogenous rodent genomic locus for the TCRa, TCRP, MHC class I, CD8a, CD8P and P2M respectively.

[0341] Preferred rodents comprise insertions at multiple of these rodent loci, with preferred combinations comprising the inserted human DNA at the TCRa, TCRP, MHC class I, CD8a, and CD8P loci; and preferably the inserted human DNA at all of the TCRa, TCRP, MHC class I , CD8a, CD8P and B2M loci. In this way there can be presentation of an antigen in the rodent by a human MHC class I polypeptide to a human TCR and human co-receptor CD8.

[0342] However it will be appreciated that a rodent and cells of the invention may comprise only some, but not all, fully human loci described herein, such as only 1, 2, 3, 4 ,5 or 6 of the rodent loci modified to express a human polypeptide(s). For example the rodent may comprise DNA encoding a human MHC class I, which may be used in combination with rodent polypeptides expressed at the other loci listed above, or which may be used in combination with chimaeric rodent - human polypeptides expressed at the other loci listed above, or a combination of both. Therefore, any 1, 2, 3, 4, 5, 6 or 7 of the following loci may be fully human; TCRa, TCRP, MHC class I (one or both H2K and H2D), CD8a, CD8P and P2M and may be used in combination with rodent and / or chimaeric (e.g. rodent-human chimaeric) versions of the polypeptides from the other loci disclosed herein.

[0343] Preferred are rodents and cells having at least the human TCRa and TCRP insertions to allow for the production of a fully human TCR in the rodent.

[0344] By way of a first example, the rodent may comprise nucleic acid expressing a chimaeric rodent-human TCRa and / or TCRP in combination with a fully human MHC class I. In other examples, the rodent may comprise nucleic acid expressing a chimaeric rodent-human TCRa and / or TCRP in combination with a fully human CD8 a and / or fully human CD8p. The rodent may comprise nucleic acid expressing a chimaeric rodent-human TCRa and / or TCRP in combination with a fully human CD8 a and / or human CD8 p and human P2M. The rodent may comprise nucleic acid expressing a chimaeric rodent- human MHC class I in combination with a fully human TCRa and / or TCRp. The rodent may comprise nucleic acid expressing a chimaeric rodent- human MHC class I in combination with a fully human CD8 a and / or CD8 p. The rodent may comprise nucleic acid expressing a chimaeric rodent- human MHC class I in combination with a fully human CD8 a and / or p and P2M. These are non-limiting examples and other combinations of fully human loci and rodent and / or chimaeric loci are possible.

[0345] Examples of chimaeric TCRa and TCRP, MHC class I and CD8a and CD8P loci are given in, for example, W02014130671, WO2014130667 and WO2016085889A1, and the specific examples of these such loci and the specific polypeptides that are generated are hereby incorporated by reference as polypeptides and modified rodent loci that may be used in the present invention in combination with one or more fully human loci / polypeptides described herein.

[0346] In one aspect a rodent containing multiple human insertions at different loci is obtained by breeding, preferably breeding to homozygosity at each of the loci having inserted human DNA. The rodent may therefore be homozygous at one, 2, 3, 4, 5 6 or all 7 of the TCRa, TCRP, MHC class I (H2D and H2K) , CD8a, CD8P and B2M loci in the rodent (depending on the number of inserted human loci).

[0347] In one aspect the rodents are fertile, as assessed for example by litter size. Litter size is preferably at least 50%, 60%, 70%, 80%, 90% and preferably at least 95% of the litter size of a wild-type rodent, preferably 100% of the litter size of a wild-type rodent.

[0348] Preferably the genetically modified rodent or rodent cell of the invention comprises:

[0349] (i) an unrearranged T cell receptor (TCR) a variable gene locus comprising at least one human Va segment and at least one human Ja segment, wherein the TCRa variable gene is operably linked to a human TCRa constant gene sequence,

[0350] (ii) an unrearranged TCRP variable gene locus comprising at least one human vp segment, at least one human DP segment, and at least one human jp segment, wherein the unrearranged TCRP variable gene is operably linked to a human TCRP constant gene sequence wherein the unrearranged human T cell variable region gene segments are capable of rearranging to form genes that encode human T cell receptor variable domains, wherein the unrearranged TCRa variable gene locus is present at an endogenous rodent TCRa gene locus and the unrearranged TCRP variable gene locus is present at an endogenous rodent TCRP gene locus,

[0351] (iii) a nucleic acid sequence encoding a human CD8a polypeptide, wherein the nucleic acid sequence encoding a human CD8a polypeptide is present at an endogenous rodent CD8a co-receptor locus,

[0352] (iv) a nucleic acid sequence encoding a human CD8P polypeptide, the nucleic acid sequence encoding a human CD8P polypeptide is present at an endogenous rodent CD8P co-receptor locus,

[0353] (v) a nucleic acid sequence encoding a human MHC class I polypeptide, and the nucleotide sequence encoding a human MHC class I polypeptide is present at an endogenous rodent MHC class I locus (vi) a nucleic acid sequence encoding a human P2 microglobulin polypeptide, which when present, is located at an endogenous rodent P2 microglobulin locus.

[0354] Functional aspects of inserted human DNA

[0355] In one aspect the rodent expresses a human CD8 protein comprising the CD8a and CD8P polypeptides and a human MHC class I polypeptide, and the human CD8 and human MHC class I can interact in the rodent.

[0356] In one aspect the rodent expresses a human TCR comprising the TCRa and TCRP polypeptides and a human MHC class I polypeptide, and the human TCR and human MHC class I can interact in the rodent.

[0357] In one aspect the rodent or rodent cell e.g. a T cell expresses a human CD8 protein comprising the CD8a and CD8P polypeptides and a human TCR comprising the TCRa and TCRP polypeptides, and the human CD8 and the human TCR can both bind to MHC class I in the rodent.

[0358] In one aspect the rodent expresses a human CD8 protein comprising the CD8a and CD8 polypeptides and expresses a human MHC class I polypeptide and expresses a human TCR comprising the TCRa and TCR polypeptides, and the human TCR and human MHC class I and human CD8 can interact in the rodent to allow presentation of antigen to the TCR by the MHC class I aided by CD8.

[0359] In one aspect the rodent expresses a repertoire of TCRs. In one aspect the rodent expresses a repertoire of both TCRa and TCRP polypeptides.

[0360] In one aspect the human variable region of the human TCR protein is capable of interacting with various proteins on the surface of the same cell or another cell. In one aspect, the human variable region of the human TCR interacts with MHC proteins (e.g., MHC class I) presenting antigens on the surface of the second cell, e.g., an antigen presenting cell (APC). In some aspects the MHC class I protein is a non-human (e.g., rodent, e.g., mouse or rat) protein. In other embodiments, the MHC class I protein is a human protein. In one aspect, the second cell, e.g., the APC, is an endogenous non-human cell expressing a human MHC molecule. In a different embodiment, the second cell is a human cell expressing a human MHC molecule.

[0361] In one aspect the human variable region of the human TCR protein is capable of interacting with an endogenous rodent MHC class II molecule on the surface of another cell e.g. an antigen presenting cell.

[0362] In one aspect the human variable region of the human TCR protein is capable of interacting with a non- polymorphic MHC molecule, for example with human or rodent CD1 or MR1.

[0363] In one aspect the human TCR receptor is able to interact with endogenous rodent molecules such as signalling molecules or portions thereof. In one aspect the human TCR is able to interact with the rodent CD3 protein complex and the CD3-zeta (^-chain) in vivo in the rodent, such that T cell activation can take place. The genome of the rodent may thus be wild type with respect to CD3 polypeptides, e.g., CD3e, CD3y, CD36 and / or CD3^. Exemplified herein is a transgenic rodent in which the CD3 protein complex is endogenous, not humanised, and it is shown that the human TCRaP functions with the rodent CD3 to successfully produce a T cell immune response to antigen (see Example 6). Alternatively, transgenic mice have been described in which one or more CD3 polypeptides, or regions thereof, are humanised, and such human CD3 transgenes may be combined with the transgenic loci of the present invention. For example, WO2016 / 085889 (Regeneron) described targeted insertion of CD3e EC domain, CD3y EC domain and CD36 EC domain at the endogenous locus by homologous recombination in mouse, generating a transgenic mouse expressing chimaeric CD3e6y polypeptides in which the extracellular domain was human and the cytoplasmic domain was endogenous mouse. Ueda et al (Scientific Reports 7:45839 2017) described replacement of mouse CD3e, CD3y and CD36 with full-length human counterparts in C57BL / 6N ESC, using cre-lox recombination, to generate a transgenic mouse expressing fully human CD3ey6 polypeptides. Others have humanised individual epitopes of the CD3 complex - for example Crespo et al (PLoS One 2021) reported a mouse in which a single murine CD3e exon (mouse exon 5) was replaced with a humanised CD3e exon (human exon 6 with 1 substitution) containing the epitope for the anti-CD3 antibody OKT3. The humanised exon encodes 11 additional amino acids compared to mouse exon 5, some of which form the binding epitope for OKT3. The humanisation of CD3 (in whole or part), in addition to the other human transgenes described herein, provides a mouse with an immune system even closer to that of the human, which is useful as a laboratory model. Advantages of the human or humanised CD3 in the animal include the ability to use antibodies to human CD3 to target the TCR-CD3 complex in the mice, for example to assess effects of candidate therapeutic molecules. However, it is notable that functional T cells, and repertoires of T cell receptors, can suitably be generated in rodents with endogenous CD3, where there is no humanisation of the CD3 complex.

[0364] CD3 polypeptides (especially CD3e, CD3y and CD36) may signal and activate T cells less efficiently when combined with human TCR polypeptides, especially the TCR alpha constant domain, compared with the corresponding endogenous mouse TCR polypeptides. This may result from a less productive interaction of CD36 (endogenous rodent CD36, or even human CD36) with human TCR Ca compared with endogenous rodent TCR Ca. Consistent with this, it has been previously reported that "murinisation" of a human TCR alpha constant region enhanced expression of TCRs by supporting preferential pairing with CD3 in human T cells (Sommermeyer & Uckert, J. Immunol. 184:6223-6231 2010).

[0365] In some embodiments, it is preferable that there be no functional expression of endogenous TCRa polypeptide in the rodent. This avoids expression of endogenous TRAC which could otherwise dominate binding to CD3 (endogenous CD3 or human CD3). In rodents with heterozyous human TCR alpha (one allele being human, and one wild type), human and endogenous TCR alpha polypeptides compete for association with CD3 (e.g., endogenous CD3), and T cells with TCR comprising endogenous TCR alpha may out-compete those with human TCR alpha, owing to the more productive TCR signalling of the former. This is avoided with homozygosity of human TRAC.

[0366] Although pairing of human TCR with CD3 may produce less efficient TCR signalling and T cell activation compared with a TCR comprising a mouse TCR alpha constant region, this could, perhaps counterintuitively, present an advantage when using the rodent as a source of high affinity human TCRs. The TCR repertoire of an individual is shaped by positive and negative selection against the individual's MHC allele and self-antigen during T cell development where a certain level of activation signalling is required for the survival (positive selection) or deletion (negative selection) of the T cell. With a slightly less efficient activation signalling, higher affinity may be needed to reach the same level of activation required by positive and negative selection, therefore resulting an up-shift in repertoire affinity distribution. Rodents with fully human TCRs, including homozygous loci expressing human TCR alpha, are actually therefore exceptionally well suited for generating high affinity TCRs for use as therapeutics, with or without humanisation of CD3. Thus, the rodent may express rodent CD3 (e.g., rodent CD36, optionally rodent CD3e, CD3y and CD36, and optionally rodent CD3^). Optionally, CD3 loci of the rodent are wild type. Alternatively, the rodent may include human CD3 transgenes (e.g., CD3e6y, preferably in single copy number or low copy number insertion).

[0367] In one aspect the rodent is a mouse having the following control regions:

[0368] TCR alpha: the enhancer 3' of the constant region (TRAC) is mouse.

[0369] TCR delta: the enhancer 5' of the constant region (TRDC) is human (e.g. from within the human BAC clone). TCR beta: the mouse enhancer originates from the region which is 3' of mouse constant region (TRBC) and 5' of mouse Vb31 in the mouse genome, but alternatively which may be the human equivalent enhancer.

[0370] MHC Class I: The enhancer 5' of H2-D1 is mouse. b2-microglobulin: The enhancer 5' of the gene and the promoter are mouse.

[0371] CD8a and CD8b: There are 4 enhancer regions (shown in Figure 3) which are mouse: E8iv, E8iii, E8ii and E8i.

[0372] Methods of making a rodent

[0373] Various methods of making rodents of the invention are described herein. These include:

[0374] A method of modifying a rodent to express a human TCRa and / or TCRP polypeptide, the method comprising (i) inserting at an endogenous TCRa locus of a rodent a nucleotide sequence encoding a human TCRa polypeptide, and / or (ii) inserting at an endogenous TCRP locus of a rodent a nucleotide sequence encoding a human TCRP polypeptide, wherein the rodent expresses a human TCR comprising the human a and / or p polypeptide(s).

[0375] A method of modifying a rodent to express a human CD8 a and / or p polypeptides comprising (i) inserting at an endogenous CD8a locus of a rodent a nucleotide sequence encoding a human CD8a polypeptide, and / or (ii) inserting at an endogenous CD8P locus of a rodent a nucleotide sequence encoding a human CD8P polypeptide, wherein the rodent expresses the human CD8a and / or CD8P polypeptide(s).

[0376] A method of modifying a rodent to express a human MHC class I polypeptide comprising (i) inserting at an endogenous rodent MHC class I locus a human MHC class I polypeptide, wherein the rodent expresses a human MHC class I polypeptide from a rodent MHC class I locus.

[0377] A method of modifying a rodent to express a human P2 microglobulin polypeptide comprising (i) inserting at an endogenous rodent P2 microglobulin locus a human P2 microglobulin polypeptide, wherein the rodent expresses a human P2 microglobulin polypeptide from a rodent P2 microglobulin locus.

[0378] A method for making a genetically modified rodent that expresses a human T cell receptor, human MHC1 and human CD8 comprising: inserting at an endogenous rodent TCRa variable gene locus an unrearranged human TCRa variable gene locus comprising at least one human Va gene segment and at least one human Ja gene segment operably linked to a human TCRa constant region; inserting at an endogenous rodent TCRP variable gene locus an unrearranged human TCRP variable gene locus comprising at least one human vP gene segment, at least one human DP gene segment, and at least one human jp gene segment operably linked to a human TCRP constant region inserting at an endogenous rodent CD8a gene locus a nucleic acid sequence encoding a human CD8a polypeptide inserting at an endogenous rodent CD8P gene locus a nucleic acid sequence encoding a human CD8P polypeptide; and optionally, inserting at an endogenous rodent P2 microglobulin gene locus a nucleic acid sequence encoding a human or chimaeric humanrodent P2 microglobulin.

[0379] The insertion of the human DNA may be accompanied by a deletion of the DNA at the insertion site, or there may be no deletion. Where a deletion of the endogenous rodent DNA takes place it may be effected at the same time as the insertion of the human DNA, or may be carried out at an earlier or later stage. Where the rodent gene or locus equivalent of the inserted human DNA, or part thereof, remains in the genome, it is inactivated or substantially inactivated, for example by inversion of the rodent locus or by a disrupting insertion into the locus or a disrupting point mutation or substitution. Where the locus is inverted, any non-TCR genes that are located within the inverted region preferably remain active and are expressed appropriately with respect to level (amount), tissue specificity (spatial) and temporal regulation.

[0380] Therefore, methods for insertion of human DNA as described herein may further include the step of the deletion of host rodent DNA at the equivalent endogenous rodent locus, preferably where that deletion causes inactivation of the host locus and prevention of the host polypeptide expression from that locus.

[0381] Methods for insertion of human DNA as described herein may further include the step of the inactivation of host rodent DNA at the equivalent endogenous rodent locus. It will be apparent that the invention also discloses rodents that not only comprise an insertion at a locus as described herein but also have inactivation of the equivalent rodent genomic locus such that no equivalent rodent polypeptide is generated.

[0382] As well as methods that refer to the insertion of human DNA herein, it will be seen that all methods therefore may extend to be methods for replacing rodent DNA with equivalent human DNA from the equivalent human locus, such that rodent DNA is deleted from the relevant locus (e.g. rodent TCRa DNA is deleted and human TCRa DNA is inserted at the TRA locus).

[0383] Methods for the large scale replacement of rodent DNA with human DNA are well known, as disclosed for example in the context of the humanisation of the mouse IG loci - see Lee et al (Lee et al, 2014 Vol 32 Number 4 April 2014 Nature Biotechnology), Macdonald et al. 2014 and Murphy et al. 2014. Such methods include recombineering of BACs to generate suitable human DNA for insertion using recombineering, followed by insertion of recombineered BACs into ES cells and then incorporation of the human DNA into the ES cell genome by e.g. recombinase mediated cassette exchange, or iterative homologous recombination, or combinations thereof.

[0384] The host DNA may be deleted if required prior to insertion of the human DNA. Such deletion may be achieved by insertion of SSR sites flanking the locus and deletion of DNA using the recombinase sites.

[0385] In one aspect the first insertion event for any locus may involve the targeted insertion of an initiation cassette, to provide a unique site within the genome for the subsequent insertion of the desired human DNA. Therefore only 1 targeting event into the genome is needed to initiate the targeted insertion of human DNA at each locus. An initiation cassette may comprise one or more SSR and / or transposases sites, which allow for iterative use of the cassette in insertion - see for example techniques disclosed in Lee et al., above.

[0386] Cells such as ES cells may be screened to assess correct insertion events using techniques well known in the art e.g. Southern blotting, PCR, quantitative PCT (e.g., real-time PCR using TAQMAN®), fluorescence in situ hybridization, Northern blotting, flow cytometry, Western blotting, immunocytochemistry, immunohistochemistry, etc.

[0387] In one aspect the invention relates to a method for making a genetically modified fertile rodent that expresses a human T cell receptor, the method comprising deletion of the rodent TCR p from at least rodent v i to Dpi, including a deletion of serine proteases genes Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2, wherein one or more or all of the deleted serine proteases genes Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2 are re-inserted into the rodent genome. In one aspect the deletion is homozygous.

[0388] The invention also relates to a method for making a fertile rodent comprising a genetic knock out of the endogenous T cell receptor beta chain polypeptide, the method comprising deletion of the rodent TCR p from at least rodent vpi to Dpi, including a deletion of serine proteases genes Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2, wherein one or more or all of the deleted serine proteases genes Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2 are re-inserted into the rodent genome.

[0389] In one aspect the deletion is homozygous.

[0390] Uses of Genetically Modified Rodents

[0391] The genetically modified rodents comprising human CD8, human MHC class I and human TCR, can present peptides to T cells in a "human-like" manner, because substantially all of the components of the complex are human. The genetically modified rodent of the invention can be used to study the function of a human immune system in the rodent, and antigens and antigen epitopes can be identified that elicit an immune response. Rodents can be used, for example, to identify T cell epitopes or human cancer epitopes, in vaccine development; identification of TCR sequences and T cells with affinity to human pathogens or cancer antigens, use in identification of TCR for use in adoptive T cell therapy; use in the evaluation of vaccine candidates and vaccination approaches; use in studying human autoimmunity; use in studying human infectious diseases.

[0392] Thus, the genetically engineered rodents of the present invention are useful, among other things, for evaluating the capacity of an antigen to initiate an immune response in a human, and for generating a diversity of antigens and identifying a specific antigen that may be used in human vaccine development.

[0393] Rodents disclosed herein comprising inserted DNA encoding human TCR can be used to generate a human TCR / TCR polypeptide, or functional fragment(s) thereof, or to generate sequence information about such TCR / TCR polypeptide and functional fragment(s) thereof, the TCR being directed to an antigen with which the rodent has been immunised.

[0394] T cell therapies are of current medical interest. In particular cytotoxic T cells can recognise and eliminate an antigen of interest, e.g., a viral or bacterial antigen, or a tumor antigen, etc., or cells that present it. Adoptive cell therapies look to isolate tumor infiltrating lymphocytes from tumor cell mass, expanding them in vitro using T cell growth factors, and transferring them back to the patient. Rodents described herein may be a source of a repertoire of human T cell receptors, e.g., high-affinity human T cell receptors for use in adoptive T cell transfer.

[0395] Methods described below for generation of a human TCR suitably comprise use of a rodent comprising inserted human DNA encoding a human TCRa polypeptide and human TCRb polypeptide. Methods for the generation or use of a human TCR equally apply to the generation of a functional part of the human TCR, suitably comprising at least the binding portion of the TCR. Preferably the binding of the TCR fragment to antigen is the same or substantially the same as the full length TCR.

[0396] Methods disclosed herein that use or generation of a TCR equally apply to the use, or generation of a functional fragment of the TCR.

[0397] In one aspect is disclosed a method of generating a T cell receptor to a human antigen comprising immunizing a rodent described herein with an antigen of interest, allowing the rodent to mount an immune response, optionally isolating from the animal an activated T cell with specificity for the antigen of interest, determining the nucleic acid sequence of a T cell receptor expressed by an antigen-specific T cell. Methods of immunising animals to generate T cell responses are known in the art. Immunisation may comprise administering a target peptide antigen of interest to the rodent in an immunisation regimen comprising a prime and at least one boost. The immunisation regimen may be a prime followed by a single boost or prime followed by two or three boosts. The peptide may be administered by subcutaneous injection for the prime and / or boost immunisations. The peptide may be combined with adjuvant for administration. Example immunisation regimens and adjuvants are indicated in the Example 6.

[0398] In one aspect antigen specific CD8 cells may be sorted with a labelled MHC multimer (e.g. tetramer or higher order multimer), and then the TCR nucleic acid in the cell sequenced to identify paired heavy and light TCR chains.

[0399] In one aspect, the invention provides a method of producing a human T cell receptor specific for an antigen of interest comprising immunizing a rodent of the invention as described herein with the antigen of interest; allowing the animal to mount an immune response; optionally isolating from the animal a T cell reactive to the antigen of interest; determining a nucleic acid sequence of the human TCR variable regions expressed by an antigen-specific T cell; and expressing nucleotide constructs encoding the human T cell receptor polypeptide chains specific for the antigen of interest. Expressing the nucleotide construct may be expression of a cell membrane expressed TCR or a soluble TCR.

[0400] In one aspect is disclosed a method of identifying a T cell with specificity against an antigen of interest, e.g., a tumor associated antigen, comprising immunizing a rodent of the invention described herein with the antigen of interest, allowing the animal to mount an immune response, and isolating from the nonhuman animal a T cell with specificity for the antigen.

[0401] In one aspect is disclosed a method for identifying a human T cell epitope, comprising exposing a rodent of the invention as described herein to an antigen comprising a putative T cell epitope, allowing the rodent to mount an immune response, isolating from the rodent an MHC I T cell that binds the epitope, and identifying the epitope bound by said T cell.

[0402] In one aspect is disclosed a method for determining whether a peptide will provoke a cellular immune response in a human, the method comprising exposing a genetically modified rodent a rodent of the invention as described herein to the peptide, allowing the rodent to mount an immune response, and detecting a CD8+ T cell comprising a human TCR that binds a sequence of the peptide presented by a human MHC class I . In one aspect is disclosed a method for determining whether a putative antigen contains an epitope that upon exposure to a human immune system will generate an HLA class I restricted immune response, comprising exposing a rodent a rodent of the invention as described herein to the antigen and measuring an antigen-specific HLA class I restricted immune response in the mouse.

[0403] The genetically engineered rodent described herein may be useful in a method for the identification of T cell receptors, e.g., high-affinity T cell receptors, that recognize an antigen of interest, e.g., a tumor or another disease antigen. A method may comprise exposing the rodent a rodent of the invention described herein to an antigen, allowing the rodent to mount an immune response to the antigen, and determining the sequence of a T cell receptor that binds the antigen presented by a human MHC I.

[0404] A genetically engineered rodent as described herein may be useful for identification of T cell receptors, e.g., high-affinity T cell receptors, that recognize an antigen of interest, e.g., a tumor or another disease antigen. The method may comprise: exposing the rodent of the invention described herein to an antigen, allowing the rodent to mount an immune response to the antigen, optionally isolating from the rodent a T cell comprising a T cell receptor that binds the antigen presented by a human MHC class I, and determining the sequence of a T cell receptor that binds the antigen.

[0405] An antigen of interest mentioned herein may be any antigen that is known to cause or be associated with a disease or condition, e.g., a tumour associated antigen; a viral antigen, a bacterial antigen. Tumour associated antigens are known and are provided in the Cancer Immunity Peptide Database (archive.cancerimmunity.org / peptidedatabase / Tcellepitopes.htm). In some embodiments of the invention, the antigen of interest is a human antigen, e.g., a human tumour associated antigen. In some embodiments, the antigen is a cell type-specific intracellular antigen, and a T cell receptor is used to direct the killing of a cell expressing the antigen.

[0406] In one aspect is disclosed a method for determining T cell activation by a putative human therapeutic, comprising exposing a rodent of the invention as described herein to a putative human therapeutic (or exposing a human MHC class I -expressing cell of such an rodent to a peptide sequence of the putative therapeutic), exposing a cell of the genetically modified rodent that displays a human MHC class I / peptide complex to a T cell comprising a human CD8 capable of binding the cell of the genetically modified animal, and measuring activation of the T cell that is induced by the peptide-displaying cell of the genetically modified animal.

[0407] The genetically modified rodents of the invention can be used to identify autoantigens of relevance to human autoimmune diseases, e.g., type I diabetes, multiple sclerosis, etc. Also, the genetically modified rodents of the invention can be used to study various aspects of human autoimmune disease, and may be utilized as autoimmune disease models.

[0408] In one aspect the invention relates to a method of producing a human T cell receptor, or receptor variable region, or an antigen binding portion of a T cell receptor, to an antigen of interest comprising:

[0409] (i) Optionally making a genetically modified rodent as disclosed herein,

[0410] (ii) immunizing a genetically modified rodent disclosed herein with the antigen of interest;

[0411] (iii) allowing the rodent to mount an immune response; (iv) determining a nucleic acid sequence of a human TCR or the sequence of a TCR variable region expressed by a T cell from the rodent reactive with the antigen of interest, optionally including a step of isolating the T cell expressing such a TCR, and either

[0412] (v) expressing the human T cell receptor, or human T cell receptor variable region, or antigen binding portion of a human T cell receptor, in a cell; and optionally further formulating the expressed human T cell receptor, or human T cell receptor variable region, or an antigen binding portion thereof, with a pharmaceutically acceptable excipient; or

[0413] (vi) inserting nucleic acid encoding the human T cell receptor, or human T cell receptor variable region, or an antigen binding portion thereof, into a cell, ex vivo or in vitro, such as into a human or animal cell, further optionally wherein the cell containing the inserted nucleic acid is formulated for delivery to a human or animal, respectively, or

[0414] (vii) optionally formulating a nucleic acid (e.g. RNA or DNA) encoding the human T cell receptor, or human T cell receptor variable region, with a suitably delivery vehicle, such as a lipid or liposome, for delivery to a patient in need thereof in vivo.

[0415] The invention also includes a method of treating an individual in need thereof, the method comprising

[0416] (i) delivery of a cell according to step (vi) above into the patient in need thereof, or

[0417] (ii) delivery of a nucleic acid (e.g. RNA or DNA) encoding the human T cell receptor, or human T cell receptor variable region according the above method, optionally formulated as in step (vii) above, into a patient in need thereof.

[0418] In one aspect is provided a method for making a human TCRa and / or TCRP, or functional fragment thereof (e.g. antigen binding portion thereof), the method comprising expressing a human TCRa and / or TCRP polypeptide(s), or functional fragment(s) thereof, in a cell from a nucleotide sequence which is obtained from or identified from a rodent as described herein, the nucleotide sequence encoding a TCR or functional fragment thereof as described herein.

[0419] In one aspect, the nucleic acid to be expressed is comprised within is a viral vector; in a specific embodiment, the viral vector is a lentiviral vector.

[0420] In one embodiment, the cell for expression of a nucleic acid (e.g. encoding a human TCR or functional fragment thereof) is selected from COS, CHO, 293, HeLa, and a retinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6™ cell).

[0421] In one aspect a cell is provided that expresses a human polypeptide having a sequence obtained from immunisation of a rodent having a human TCR as described herein.

[0422] TCR alpha and beta variable domains isolated from rodents described herein may be recombinantly produced in the context of soluble molecules including human TCR constant regions. TCR variable domain sequences according to the present invention are particularly well suited for development of soluble TCR therapeutics for use in human patients, including bispecific T-cell engagers, in formats in which the variable domain is linked to a human TCR constant domain. As reported herein (Example 8), 100 % (124 / 124) of tested TCR clonotypes were recombinantly expressable in soluble form. No clonotypes were encountered that failed to express in soluble form with human constant regions. The good expression levels and solubility of TCR binding fragments observed here may be attributable, at least in part, to being species matched - fully human - as it is known that the interaction between the variable and constant regions of mouse TCRs is important for folding. As TCRs in rodents of the present invention are fully human, selection of T cells for antigen-binding occurs in vivo in the context of a human TCR variable region linked to a human TCR constant region. Conversion of a selected TCR to a soluble format, in which the isolated TCR variable region is linked to a human TCR constant region, therefore reflects the context in which the TCRs were selected in vivo. In contrast, the transgenic "VelociT" mice described by Moore et al., Sci Immunol 6 2021 produced TCRs with human variable domains and mouse constant domains. TCRs from VelociT mice require reformatting to replace the mouse constant domain with a human constant domain, to reduce immunogenicity in human patients. Such reformatting of a chimaeric TCR to change the constant region could potentially reduce or abrogate its expression, as the human variable region was not originally selected in vivo in the context of a fully human TCR.

[0423] Cells and tissues

[0424] In one aspect the invention relates to cells, e.g., T cells, or APCs derived from the rodents described herein. The invention also relates to tissues and embryos derived from the rodents described herein.

[0425] In one aspect a cell isolated from a rodent as described herein is an ES cell or hematopoietic stem cell other cell capable of contributing to tissues and organs of a non-human mammal. The cell may be an iPS cell generated from a cell obtained from the rodent.

[0426] In one aspect, the isolated cell is a T cell. In one aspect the T cell expresses a human TCRa and / or TCRP, and / or the T cell expresses human CD8a and / or CD8p. In one aspect the T cell is a CD8+ T cell.

[0427] In one aspect, the isolated cell is an antigen presenting cell. In one aspect the antigen presenting cell expresses a human MHC class I and / or human P2M molecule.

[0428] Modification by manipulation of ES cells, such as mouse or rat ES cells is well known in the art. The invention also extends to modification of hematopoietic stem cell or other cell capable of contributing to tissues and organs of a non-human mammal, preferably which is able to produce a repertoire of human TCRs.

[0429] In one aspect the invention relates to a pluripotent cell comprising an unrearranged human TCR locus encoding a human TCRa and p polypeptide as described herein. In one embodiment, the induced pluripotent cell is derived from a rodent described herein.

[0430] The invention also relates to a cell line which is grown from or otherwise derived from a cell as described herein. In one aspect the cell line is an immortalised cell line.

[0431] The cell line may comprise rearranged or unrearranged VDJ or VJ genes from a TCRa and / or TCRP polypeptide. The cell may be immortalised by fusion to a tumour cell to provide an TCR producing cell and cell line, or be made by direct cellular immortalisation.

[0432] In one aspect, a hybridoma or quadroma is provided, derived from a cell of a rodent as described herein.

[0433] Development of a rodent from the rodent ES cell is well known in the art, and comprises the steps of injection of the ES cell into a blastocyst, followed by implantation of chimaeric blastocystys into females to produce offspring which can be bred and selected for the required insertion.

[0434] In one aspect the invention relates to a chimeric rodent comprised of ES cell-derived tissue and host embryo derived tissue.

[0435] In one aspect the invention relates to a rodent embryo wherein the embryo comprises a donor ES cell that is derived from a rodent, as described herein. In one aspect, the embryo comprises an ES donor cell that comprises an unrearranged human TCR locus, and host embryo cells.

[0436] In one aspect is provided is a tissue, wherein the tissue is derived from a rodent as described herein, and expresses a humanTCR polypeptide (e.g., TCRa and / or TCRP).

[0437] Rodent strains

[0438] Preferred rodents are rats or mice. Mice are especially preferred.

[0439] In one aspect a mouse may be a BL6 strain, such as C57BL strain, e.g. a strain selected from C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / Ola. In another aspect the mouse may be a 129 strain, e.g. selected from the group consisting of a strain that is 129P1, 129P2, 129P3, 129X1 , 129S1 (e.g., 129S1 / SV, 129Sl / Svlm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1 , 129T2.

[0440] In one aspect the genetically modified mouse is a mix of a 129 strain and a C57BL / 6 strain. In another specific aspect, the mouse is a mix of 129 strains, or a mix of BL / 6 strains. In a specific embodiment, the 129 strain of the mix is a 129S6 (129 / SvEvTac) strain.

[0441] In another embodiment, the mouse is a BALB strain, e.g., BALB / c strain. In yet another embodiment, the mouse is a mix of a BALB strain and an aforementioned strain.

[0442] Where the rodent is a rat, the rat may be selected from a Wistar rat, an LEA strain, a Sprague Dawley strain, a Fischer strain, F344, F6, and Dark Agouti. In one embodiment, the rat strain is a mix of two or more strains selected from this group of strains.

[0443] The invention relates to any mouse or rat strain above comprising any genetic modification disclosed herein. Cell development / T cell development

[0444] The insertion of human DNA at an endogenous rodent locus suitably does not impact the ability of the relevant rodent cells to develop normally, for example, to be able to participate in presentation of MHC class I bound antigen and / or TCR recognition of such a bound antigen.

[0445] In one aspect the rodents and rodent cells described herein comprise a genetic modification that does not alter proper lineage choice and development of T cells.

[0446] In one aspect there is proper spatial and temporal protein expression of inserted human DNA.

[0447] In one aspect the rodents and rodent T cells described herein comprising human TCRa and / or TCR produce T cells that are capable of undergoing thymic development, progressing from DN1 to DN2 to DN3 to DN4 to DP and to CD8 SP T cells. Such T cells of the rodent of the invention express cell surface molecules typically produced by a T cell during a particular stage of thymic development (e.g., CD25, CD44, Kit, CD3, pTcc, etc.). Thus, the rodents described herein express pTa complexed with TCRa at the DN3 stage of thymic development. The non-human animals described herein express T cells capable of undergoing thymic development to produce CD8+ T cells. In one aspect the rodents produce T cells that are capable of undergoing normal T cell differentiation in the periphery. In some embodiments, the non- human animals described herein are capable of producing a normal repertoire of effector T cells, e.g., CTL (cytotoxic T lymphocytes), TH1 , TH2, Treg etc.

[0448] In one aspect the rodents and rodent T cells described herein comprising human CD8a and / or CD8 polypeptides produce T cells that are capable of undergoing thymic development, progressing from DN1 to DN2 to DN3 to DN4 to DP and to CD8 SP T cells. Such T cells of the rodent of the invention express cell surface molecules typically produced by a T cell during a particular stage of thymic development (e.g., CD25, CD44, Kit, CD3, pTcc, etc.). Thus, the rodents described herein express pTa complexed with TCRa at the DN3 stage of thymic development. The non-human animals described herein generate T cells capable of undergoing thymic development to produce CD8+ T cells. In one aspect the rodents produce T cells that are capable of undergoing normal T cell differentiation in the periphery. In some embodiments, the non-human animals described herein are capable of producing a normal repertoire of effector T cells, e.g., CTL (cytotoxic T lymphocytes), TH1 , TH2, Treg etc. In one aspect the CD8 locus shows proper CD8a and / or expression (proper spatial and temporal protein expression), CD8+ T cell development, CD8 lineage choice, and co-receptor function.

[0449] In one aspect the rodents described herein comprising a human MHC class I polypeptide are able to produce antigen presenting cells expressing MHC class I that are able to develop essentially as normal, and at least to present antigen to a TCR, such as a human TCR.

[0450] General features of the invention

[0451] General features of the invention are separably combinable with all aspects of the disclosure.

[0452] In one aspect one or more or all of the inserted fully human sequences are operably linked to one or more endogenous rodent control sequences, such as promoter and / or enhancers. In one aspect the orientation of the inserted human DNA with respect to the centromere is the same as the equivalent endogenous rodent locus and the position of the inserted human DNA with respect to the centromere is the same or substantially the same as that of the equivalent endogenous rodent locus (e.g. the human TRA insertion is made in the same orientation as the rodent TRA and in the same or substantially the same position, and at the endogenous locus.

[0453] In one aspect variation is permitted in the inserted human sequences. For example, human TCRa TCRP MHC class I, CD8a and / or CD8 polypeptide may comprise one or more conservative or non-conservative modification(s). Therefore a rodent is provided that expresses a human TCRa and / or p polypeptide sequence, CD8a and / or p polypeptide sequence, MHC class I or P2M, wherein the human polypeptide sequence is at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a human TCRa, TCRP CD8a, CD8P, MHC class I or P2M, polypeptide sequence respectively.

[0454] In another option the human polypeptide sequence is at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a human TCRa, TCRP CD8a, CD8P, MHC class I or P2M, polypeptide sequence respectively over a functional portion of the polypeptide or protein complex - such as a domain of a polypeptide or protein.

[0455] In one embodiment, the human polypeptide sequence comprises one or more conservative substitutions. A conservative substitution includes substitution of an amino acid residue by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Conservative amino acid substitutions may be achieved by modifying a nucleotide sequence so as to introduce a nucleotide change that will encode the conservative substitution. In general, a conservative amino acid substitution will not substantially change the functional properties of interest of a protein, for example, the ability of MHC class 1 to present a peptide of interest. Examples of groups of amino acids that have side chains with similar chemical properties include aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxyl side chains such as serine and threonine; amide-containing side chains such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, arginine, and histidine; acidic side chains such as aspartic acid and glutamic acid; and, sulphur-containing side chains such as cysteine and methionine. Conservative amino acids substitution groups include, for example, valine / leucine / isoleucine, phenylalanine / tyrosine, lysine / arginine, alanine / valine, glutamate / aspartate, and asparagine / glutamine. In addition to the nucleic acid residues encoding a human MHC class I polypeptide described herein due to the degeneracy of the genetic code, other nucleic acids may encode the disclosed polypeptides. Therefore, in addition to a genetically modified non-human animal that comprises in its genome a nucleotide sequence encoding MHC class I polypeptides with conservative amino acid substitutions, a non-human animal whose genome comprises a nucleotide sequence that differs from that described herein due to the degeneracy of the genetic code is also provided by the present disclosure.

[0456] In one aspect the human DNA inserted into the rodent genome is human genomic DNA.

[0457] In another aspect the human DNA inserted into the rodent genome may be in the form of non -genomic DNA, for example, where the human TCR loci are inserted, the DNA may be in the form of a minigene in which elements of the human genomic DNA have been deleted to reduce the insert size compared with the genomic human DNA. For example non-coding sequences or non-functional sequences may be deleted. Where multiple DNA fragments are inserted, inserted human DNA may be contiguous or noncontiguous. Inserted human DNA preferably is in the form of contiguous gene or contiguous human locus within the rodent genome.

[0458] Human polypeptides described herein may be expressed under the control of endogenous non-human regulatory elements, such as rodent regulatory elements, e.g. promoters, enhancers or silencers. These elements may be the naturally occurring rodent regulatory element at the natural position in the rodent, in particular where the element has not been removed from the genome or repositioned (even though its position in respect of other rodent genetic elements may have changed relatively speaking due to e.g. insertion of human DNA. The human polypeptides described herein may alternatively be expressed under the control of human regulatory elements, such as human regulatory elements, e.g. promoters, enhancers or silencers.

[0459] In one aspect the inserted loci are regulated, individually or collectively, by at least one human regulatory element and at least one endogenous rodent regulatory element. For example, there may be use of human promoter(s) and host enhancer sequences and one or more loci.

[0460] Reference to a "rodent comprising" or a "rodent cell comprising" herein is a reference to the genome of the rodent or rodent cell comprising the specific nucleic acid or DNA, unless otherwise specified.

[0461] The term "operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. As such, a nucleic acid sequence encoding a protein may be operably linked to regulatory sequences (e.g., promoter, enhancer, silencer sequence, etc.) so as to retain proper transcriptional regulation. In addition, various portions of a human protein may be operably linked to retain proper folding, processing, targeting, expression, and other functional properties of the protein in the cell. Unless stated otherwise, various domains of the human protein are operably linked to each other.

[0462] "Functional" as used herein, e.g., in reference to a functional polypeptide, refers to a polypeptide that retains at least one biological activity normally associated with the native protein. A replacement at an endogenous locus (e.g., replacement at an endogenous non-human MHC locus) results in a locus that fails to express a functional endogenous polypeptide, e.g., MHC class I polypeptide. Likewise, the term "functional" as used herein in reference to functional portion of a protein, refers to an extracellular domain that retains its functionality, e.g., in the case of a functional portion of a TCR, the ability to bind an antigen on a MHC class I.

[0463] As mentioned above, the rodent of the invention preferably does not express a functional rodent polypeptide from the modified endogenous locus. For example, the rodent does not express rodent TCRa polypeptide from the endogenous TRA locus where DNA encoding the human TCRa polypeptide has been inserted into the endogenous locus . For example, the endogenous locus may be replaced before, or concomitant with, or after the insertion of the human DNA at the locus. Alternatively the endogenous locus may be inactivated e.g. by mutation or inversion, such that there is no expression of the rodent polypeptide. Reference herein to an endogenous gene locus is the rodent genomic locus comprising the coding region(s), any intervening DNA , such as introns, and promoter regions. In one aspect the inserted human DNA replaces all or a part of the host locus. In one aspect the host locus may be retained but is displaced upstream or downstream by the insertion of the human DNA at the endogenous locus. Endogenous gene expression from the endogenous locus is preferably inactivated however, in one aspect, essential genes which do not encode any of the TCR / MHC class I, CD8 or B2M may be retained or reinserted. For example, non-TCR related genes related to fertility may be retained in the TCRP locus, such as genes encoding one or more serine proteases located in the TCR locus

[0464] In one aspect the TCR locus or TCR gene locus herein, refers to the genomic DNA comprising the TCR coding region, including the entire TCR coding region, including unrearranged V(D)J sequences, promoters, enhancer sequence(s), constant sequence(s), and any upstream or downstream (UTR, regulatory regions, etc.), or intervening DNA sequence (introns, etc.). The TCR variable locus or TCR variable gene locus refers to genomic DNA comprising the region that includes TCR variable region segments (V(D)J region) but excludes TCR constant sequences.

[0465] It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine study, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims. All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention [pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and / or." Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0466] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0467] The term "or combinations thereof' as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, SAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context. Various uses for TCRs made in mice are disclosed in EP2958937B1, EP2771357B1, and EP2958938B1, and these disclosures are specifically incorporated by reference herein, but in the context of rodents containing fully human loci as disclosed herein.

[0468] The following detailed description includes exemplary representations of various embodiments of the invention, which are not restrictive of the invention as claimed. The accompanying figures constitute a part of this specification and, together with the description, serve only to illustrate embodiments and not to limit the invention.

[0469] The following numbered clauses represent embodiments of the invention.

[0470] 1 A genetically modified rodent, comprising:

[0471] (i) an unrearranged T cell receptor (TCR) a variable gene locus comprising at least one human Va segment and at least one human Ja segment, wherein the TCRa variable gene is operably linked to a human TCRa constant gene sequence,

[0472] (ii) an unrearranged TCRP variable gene locus comprising at least one human vp segment, at least one human DP segment, and at least one human jp segment, wherein the unrearranged TCRP variable gene is operably linked to a human TCRP constant gene sequence wherein the unrearranged human T cell variable region gene segments are capable of rearranging to form genes that encode human T cell receptor variable domains, wherein the unrearranged TCRa variable gene locus is present at an endogenous rodent TCRa gene locus and the unrearranged TCRP variable gene locus is present at an endogenous rodent TCRP gene locus.

[0473] 2. A rodent according to clause 1, comprising a genome encoding an MHC class I polypeptide in which the al and a2 domains are human.

[0474] 3. A rodent according to clause 2 wherein the MHC class I polypeptide comprises an a3 domain that binds CD8 expressed in the rodent.

[0475] 4. A rodent according to clause 3, wherein the a3 domain is human and the CD8 comprises a human MHC class I binding domain.

[0476] 5. A rodent according to clause 4, wherein the CD8 comprises human CD8a.

[0477] 6. A rodent according to clause 4, wherein the CD8 comprises human-rodent chimaeric CD8a.

[0478] 7. A rodent according to any of clauses 4 to 6, wherein the CD8 comprises human CD8p.

[0479] 8. A rodent according to any of clauses 4 to 6, wherein the CD8 comprises human-rodent chimaeric CD8p.

[0480] 9. A rodent according to any of clauses 5 to 8, comprising a genome in which the human or human-rodent chimaeric CD8 are present at an endogenous rodent CD8 locus. 10. A rodent according to any of clauses 2 to 9, wherein the MHC class I polypeptide comprises human transmembrane and cytoplasmic domains.

[0481] 11. A rodent according to clause 3, wherein the MHC class 1 a3 domain is rodent.

[0482] 12. A rodent according to clause 11, wherein the CD8 comprises endogenous rodent CD8.

[0483] 13. A rodent according to any of clauses 2 to 12, wherein the DNA encoding the MHC class I polypeptide is integrated at the endogenous rodent MHC class I locus.

[0484] 14 A genetically modified rodent according to clause 1 wherein then rodent genome additionally comprises a nucleic acid sequence encoding an MHC class I polypeptide, wherein the MHC class I is (i) fully human, or is (ii) the endogenous rodent MHC class I, or is (iii) a chimaeric human-rodent MHC class I, for example is a chimaeric MHC class I that comprises a rodent alpha 3 domain capable of binding to a rodent CD8 protein, optionally wherein the nucleic acid sequence encoding an MHC class I polypeptide is present at an endogenous locus

[0485] 15. A genetically modified rodent according to any one of clauses 1 or 14 wherein the rodent genome additionally comprises a nucleic acid sequence encoding a CD8a polypeptide at an endogenous rodent CD8a co-receptor locus, and a nucleic acid sequence encoding a CD8P polypeptide at an endogenous rodent CD8P co-receptor locus, optionally wherein the nucleic acid sequences are selected from:

[0486] (i) A nucleic acid sequence encoding a human CD8a polypeptide, wherein the nucleic acid sequence encoding a human CD8a polypeptide is present at an endogenous rodent CD8a co-receptor locus, and a nucleic acid sequence encoding a human CD8P polypeptide, the nucleic acid sequence encoding a human CD8P polypeptide is present at an endogenous rodent CD8P co-receptor locus;

[0487] (ii) A nucleic acid sequence encoding a chimaeric human-rodent CD8a polypeptide, wherein the nucleic acid sequence encoding a chimaeric human-rodent CD8a polypeptide is present at an endogenous rodent CD8a co-receptor locus, a nucleic acid sequence encoding a chimaeric humanrodent CD8P polypeptide, the nucleic acid sequence encoding a chimaeric human-rodent CD8P polypeptide is present at an endogenous rodent CD8P co-receptor locus; and

[0488] (iii) a nucleic acid sequence encoding a rodent CD8a polypeptide at the endogenous locus and a nucleic acid sequence encoding a rodent CD8 p polypeptide at the endogenous locus.

[0489] 16. A genetically modified rodent according to any one of clauses 1, 14 or 15 wherein then rodent genome comprises a nucleic acid sequence encoding a human P2 microglobulin polypeptide.

[0490] 17. A genetically modified rodent according to clauses 1, and any one of 14-16 wherein the rodent genome comprises a nucleic acid sequence encoding a human P2 microglobulin polypeptide, optionally which is located at an endogenous rodent P2 microglobulin locus.

[0491] 18. A genetically modified rodent according to clause 1, comprising: (i) an unrearranged T cell receptor (TCR) a variable gene locus comprising at least one human Va segment and at least one human Ja segment, wherein the TCRa variable gene is operably linked to a human TCRa constant gene sequence,

[0492] (ii) an unrearranged TCRP variable gene locus comprising at least one human vp segment, at least one human DP segment, and at least one human jp segment, wherein the unrearranged TCRP variable gene is operably linked to a human TCRP constant gene sequence wherein the unrearranged human T cell variable region gene segments are capable of rearranging to form genes that encode human T cell receptor variable domains, wherein the unrearranged TCRa variable gene locus is present at an endogenous rodent TCRa gene locus and the unrearranged TCRP variable gene locus is present at an endogenous rodent TCRP gene locus and wherein the rodent genome comprises either

[0493] [A] a nucleic acid sequence encoding a human CD8a polypeptide, wherein the nucleic acid sequence encoding a human CD8a polypeptide is present at an endogenous rodent CD8a co-receptor locus, a nucleic acid sequence encoding a human CD8P polypeptide, the nucleic acid sequence encoding a human CD8P polypeptide is present at an endogenous rodent CD8P co-receptor locus; and a nucleic acid sequence encoding a human MHC class I polypeptide present at an endogenous rodent MHC class I locus; or

[0494] [B] a nucleic acid sequence encoding a chimaeric human-rodent CD8a polypeptide, wherein the nucleic acid sequence encoding a chimaeric human-rodent CD8a polypeptide is present at an endogenous rodent CD8a co-receptor locus, a nucleic acid sequence encoding a chimaeric humanrodent CD8P polypeptide, the nucleic acid sequence encoding a chimaeric human-rodent CD8P polypeptide is present at an endogenous rodent CD8P co-receptor locus, and a nucleic acid sequence encoding a human MHC class I polypeptide present at an endogenous rodent MHC class I locus; wherein the chimaeric CD8 protein is capable of binding to the human MHC class I; or

[0495] [C] a nucleic acid sequence encoding a rodent CD8a polypeptide at the endogenous locus and a nucleic acid sequence encoding a rodent CD8 p polypeptide at the endogenous locus; and a nucleic acid sequence encoding a chimaeric human-rodent MHC class I polypeptide present at an endogenous rodent MHC class I locus, wherein the rodent CD8 protein is capable of binding to the chimaeric MHC class I, optionally wherein then rodent genome comprises a nucleic acid sequence encoding a human P2 microglobulin polypeptide which, when present, is located at an endogenous rodent P2 microglobulin locus

[0496] 19. A genetically modified rodent cell, such as an ES cell, comprising: (i) an unrearranged T cell receptor (TCR) a variable gene locus comprising at least one human Va segment and at least one human Ja segment, wherein the TCRa variable gene is operably linked to a human TCRa constant gene sequence,

[0497] (ii) an unrearranged TCRP variable gene locus comprising at least one human vp segment, at least one human DP segment, and at least one human jp segment, wherein the unrearranged TCRP variable gene is operably linked to a human TCRP constant gene sequence wherein the unrearranged human T cell variable region gene segments are capable of rearranging to form genes that encode human T cell receptor variable domains, wherein the unrearranged TCRa variable gene locus is present at an endogenous rodent TCRa gene locus and the unrearranged TCRP variable gene locus is present at an endogenous rodent TCRP gene locus,

[0498] 20. A genetically modified rodent cell according to clause 19 wherein then rodent genome additionally comprises a nucleic acid sequence encoding an MHC class I polypeptide present at an endogenous rodent MHC class I locus, wherein the MHC class I is (i) fully human, or is (ii) the endogenous rodent MHC class I, or is (iii) a chimaeric human-rodent MHC class I, for example is a chimaeric MHC class I that comprises a rodent alpha 3 domain capable of binding to a rodent CD8 protein.

[0499] 21. A genetically modified rodent cell according to any one of clauses 19 or 20 wherein the rodent genome additionally comprises a nucleic acid sequence encoding a CD8a polypeptide at an endogenous rodent CD8a co-receptor locus, and a nucleic acid sequence encoding a CD8P polypeptide at an endogenous rodent CD8P co-receptor locus, optionally wherein the nucleic acid sequences are selected from:

[0500] (i) A nucleic acid sequence encoding a human CD8a polypeptide, wherein the nucleic acid sequence encoding a human CD8a polypeptide is present at an endogenous rodent CD8a co-receptor locus, and a nucleic acid sequence encoding a human CD8P polypeptide, the nucleic acid sequence encoding a human CD8P polypeptide is present at an endogenous rodent CD8P co-receptor locus;

[0501] (ii) A nucleic acid sequence encoding a chimaeric human-rodent CD8a polypeptide, wherein the nucleic acid sequence encoding a chimaeric human-rodent CD8a polypeptide is present at an endogenous rodent CD8a co-receptor locus, a nucleic acid sequence encoding a chimaeric humanrodent CD8P polypeptide, the nucleic acid sequence encoding a chimaeric human-rodent CD8P polypeptide is present at an endogenous rodent CD8P co-receptor locus; and

[0502] (iii) a nucleic acid sequence encoding a rodent CD8a polypeptide at the endogenous locus and a nucleic acid sequence encoding a rodent CD8 p polypeptide at the endogenous locus.

[0503] 22. A genetically modified rodent cell according to any one of clauses 19-21 wherein then rodent genome comprises a nucleic acid sequence encoding a human P2 microglobulin polypeptide, optionally which is located at an endogenous rodent P2 microglobulin locus.

[0504] 23. A genetically modified rodent cell according to clause 19, comprising: (i) an unrearranged T cell receptor (TCR) a variable gene locus comprising at least one human Va segment and at least one human Ja segment, wherein the TCRa variable gene is operably linked to a human TCRa constant gene sequence,

[0505] (ii) an unrearranged TCRP variable gene locus comprising at least one human vp segment, at least one human DP segment, and at least one human jp segment, wherein the unrearranged TCRP variable gene is operably linked to a human TCRP constant gene sequence wherein the unrearranged human T cell variable region gene segments are capable of rearranging to form genes that encode human T cell receptor variable domains, wherein the unrearranged TCRa variable gene locus is present at an endogenous rodent TCRa gene locus and the unrearranged TCRP variable gene locus is present at an endogenous rodent TCRP gene locus and wherein the rodent genome comprises either

[0506] [A] a nucleic acid sequence encoding a human CD8a polypeptide, wherein the nucleic acid sequence encoding a human CD8a polypeptide is present at an endogenous rodent CD8a co-receptor locus, a nucleic acid sequence encoding a human CD8P polypeptide, the nucleic acid sequence encoding a human CD8P polypeptide is present at an endogenous rodent CD8P co-receptor locus; and a nucleic acid sequence encoding a human MHC class I polypeptide present at an endogenous rodent MHC class I locus; or

[0507] [B] a nucleic acid sequence encoding a chimaeric human-rodent CD8a polypeptide, wherein the nucleic acid sequence encoding a chimaeric human-rodent CD8a polypeptide is present at an endogenous rodent CD8a co-receptor locus, a nucleic acid sequence encoding a chimaeric humanrodent CD8P polypeptide, the nucleic acid sequence encoding a chimaeric human-rodent CD8P polypeptide is present at an endogenous rodent CD8P co-receptor locus, and a nucleic acid sequence encoding a human MHC class I polypeptide present at an endogenous rodent MHC class I locus; wherein the chimaeric CD8 protein is capable of binding to the human MHC class I; or

[0508] [C] a nucleic acid sequence encoding a rodent CD8a polypeptide at the endogenous locus and a nucleic acid sequence encoding a rodent CD8 p polypeptide at the endogenous locus; and a nucleic acid sequence encoding a chimaeric human-rodent MHC class I polypeptide present at an endogenous rodent MHC class I locus, wherein the rodent CD8 protein is capable of binding to the chimaeric MHC class I, optionally wherein then rodent genome comprises a nucleic acid sequence encoding a human P2 microglobulin polypeptide which, when present, is located at an endogenous rodent P2 microglobulin locus.

[0509] 24. A genetically modified rodent T cell, comprising: (i) an unrearranged T cell receptor (TCR) a variable gene locus comprising at least one human Va segment and at least one human Ja segment, wherein the TCRa variable gene is operably linked to a human TCRa constant gene sequence,

[0510] (ii) an unrearranged TCRP variable gene locus comprising at least one human vp segment, at least one human DP segment, and at least one human jp segment, wherein the unrearranged TCRP variable gene is operably linked to a human TCRP constant gene sequence wherein the unrearranged human T cell variable region gene segments are capable of rearranging to form genes that encode human T cell receptor variable domains, wherein the unrearranged TCRa variable gene locus is present at an endogenous rodent TCRa gene locus and the unrearranged TCRP variable gene locus is present at an endogenous rodent TCRP gene locus.

[0511] 25. A genetically modified rodent T cell according to clause 24 additionally comprising either

[0512] [A] a nucleic acid sequence encoding a human CD8a polypeptide, wherein the nucleic acid sequence encoding a human CD8a polypeptide is present at an endogenous rodent CD8a co-receptor locus, a nucleic acid sequence encoding a human CD8P polypeptide, the nucleic acid sequence encoding a human CD8P polypeptide is present at an endogenous rodent CD8P co-receptor locus; or

[0513] [B] a nucleic acid sequence encoding a chimaeric human-rodent CD8a polypeptide, wherein the nucleic acid sequence encoding a chimaeric human-rodent CD8a polypeptide is present at an endogenous rodent CD8a co-receptor locus, a nucleic acid sequence encoding a chimaeric humanrodent CD8P polypeptide, the nucleic acid sequence encoding a chimaeric human-rodent CD8P polypeptide is present at an endogenous rodent CD8P co-receptor locus; or

[0514] [C] a nucleic acid sequence encoding a rodent CD8a polypeptide at the endogenous locus and a nucleic acid sequence encoding a rodent CD8 p polypeptide at the endogenous locus; wherein the cell expresses either

[0515] [A] human TCR and human CD8 polypeptides; or

[0516] [B] human TCR and chimaeric CD8 polypeptides; or

[0517] [C] human TCR and rodent CD8 polypeptides.

[0518] 26. A genetically modified rodent antigen presenting cell comprising: either (i) a nucleic acid sequence encoding a human MHC class I polypeptide present at an endogenous rodent MHC class I locus, or

[0519] (ii) a nucleic acid sequence encoding a chimaeric human-rodent MHC class I polypeptide present at an endogenous rodent MHC class I locus, suitably wherein the nucleic acid sequence encoding the chimaeric MHC class I has a rodent exon 4 and exons 1-3 and 5-7 are human; and

[0520] (iii) optionally, a nucleic acid sequence encoding a human P2 microglobulin polypeptide, which when present, is located at an endogenous rodent P2 microglobulin locus, wherein the cell expresses either the human MHC class I or the chimaeric MHC class I, and optionally human P2M polypeptides.

[0521] 27. A genetically modified rodent or rodent cell according to any one of clauses 1-26 wherein the endogenous locus which is modified to comprise DNA encoding a human or chimaeric polypeptide does not express the equivalent rodent polypeptide.

[0522] 28. A genetically modified rodent or rodent cell according to any one of clauses 1-27 whichdoes not express a functional endogenous TCRa polypeptide from the endogenous TCRa variable gene locus.

[0523] 29. A genetically modified rodent or rodent cell according to clause 28, which is homozygous at the TCRa gene locus.

[0524] 30. A genetically modified rodent or rodent cell according to any of clauses 1 to 29, which does not express a functional endogenous TCRP polypeptide from the endogenous TCRP variable gene locus.

[0525] 31. A genetically modified rodent or rodent cell according to clause 30, which is homozygous at the TCRP gene locus.

[0526] 32. A genetically modified rodent or rodent cell according to any one of clauses 1-31 which does not express a functional endogenous rodent CD8 co-receptor from the endogenous CD8 locus when the rodent or cell expresses a fully human MHC class I.

[0527] 33. A genetically modified rodent or rodent cell according to any one of clauses 1-32 which does not express a functional endogenous rodent MHC class I polypeptide from the endogenous MHC class I locus.

[0528] 34. A genetically modified rodent or rodent cell according to any one of clauses 1-33 which does not express a functional endogenous rodent P2 microglobulin from the endogenous rodent P2 microglobulin locus when the rodent or rodent cell comprises a nucleic acid sequence encoding a human P2 microglobulin.

[0529] 35. A genetically modified rodent or rodent cell according to any one of clauses 28-34, wherein the lack of the functional endogenous rodent TCRa variable locus comprises a deletion selected from the group consisting of (a) a deletion of all endogenous Va gene segments, (b) a deletion of all endogenous Ja gene segments, and (c) a combination thereof and / or wherein the lack of the functional endogenous rodent TCRP variable locus comprises a deletion selected from the group consisting of (a) a deletion of all endogenous vp gene segments, (b) a deletion of all endogenous DP gene segments, (c) a deletion of all endogenous jp gene segments, and (d) a combination thereof.

[0530] 36. A genetically modified rodent or rodent cell according to any one of clauses 1-35, wherein the endogenous rodent TCRa locus is deleted from the rodent genome from the distal 3' V (Vai) to the rodent 3' constant region TCRaC, inclusive, leaving the rodent enhancer in the rodent genome downstream of the TCRaC operably linked to the unrearranged human T cell receptor (TCR) a variable gene locus.

[0531] 37. A genetically modified rodent or rodent cell according to any preceding clause, wherein the human Va and Ja segments rearrange to form a rearranged human Va / Ja sequence and the human vp, DP, and jp segments rearrange to form a rearranged human vp / DP / jp sequence.

[0532] 38. A genetically modified rodent or rodent cell according to any preceding clause, wherein the rodent or rodent cell expresses a human T cell receptor comprising a human TCRa and TCRP variable domain on the surface of a T cell.

[0533] 39. A genetically modified rodent or rodent cell according to any preceding clause, wherein the unrearranged TCRa variable gene locus comprises a complete repertoire of human Ja segments and a complete repertoire of human Va segments and / or the unrearranged TCRP variable gene locus comprises a complete repertoire of human jp segments, a complete repertoire of human DP segments, and a complete repertoire of human vp segments.

[0534] 40. A genetically modified rodent or rodent cell which is either

[0535] (i) a rodent or rodent cell according to any preceding clause, wherein the rodent retains an endogenous TCRa variable gene locus and / or an endogenous rodent TCRP variable gene locus, and wherein any retained endogenous rodent TCRa variable gene locus is a non-functional locus and / or wherein any retained endogenous rodent TCRP variable gene locus is a non-functional locus, optionally wherein all or a part of the retained endogenous rodent TCRa and / or p variable gene locus is inverted with respect to its normal orientation in the rodent genome; or

[0536] (ii) A fertile rodent or rodent cell wherein the rodent TCRP locus from at least rodent vpi to Dpi is inverted within the rodent genome with respect to the natural orientation in the rodent genome, optionally the method further comprising the insertion into a rodent genome of nucleic acid, such as DNA, encoding all or a part of a TCRP from another species which is different from the rodent, such as inserting human DNA encoding all or a part of TCRp.

[0537] 41. A genetically modified male rodent which is either

[0538] (i) a rodent according to any preceding clause having substantially wild type fertility, wherein the rodent TCRP locus is deleted from at least rodent vpi to Dpi, and the genome additionally comprises one or more or all of the deleted serine proteases genes Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2 (ii) a rodent having substantially wild type fertility, wherein the rodent TCRP locus has been deleted from at least rodent vpi to Dpi, and the genome additionally comprises one or more or all of the deleted rodent serine proteases genes Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2, wherein the rodent genome is not a wild type rodent genome, and / or does not have a wild type genome at the TCRP locus, and / or cannot make a rodent TCRP chain .

[0539] 42. A genetically modified rodent or rodent cell according to any preceding clause, wherein the rodent retains the endogenous rodent TCRP variable gene locus, and wherein the endogenous rodent TCRP variable gene locus is a non-functional locus as a result of inversion of all or a part of the locus within the genome.

[0540] 43. A genetically modified rodent or rodent cell according to any preceding clause, wherein the unrearranged TCRa variable gene locus replaces all or a portion of an endogenous rodent TCRa gene locus and / or wherein the unrearranged TCRP variable gene locus replaces all or a portion of an endogenous rodent TCRP gene locus.

[0541] 44. A genetically modified rodent or rodent cell according to any preceding clause, wherein the rodent further comprises a repertoire of unrearranged human TCR6 variable region segments at the rodent TCR a locus, optionally one or more human TCR6 V gene segments, one or more human TCR6 D gene segments and one or more human TCR6 J gene segments which can be rearranged to form a human TCRD variable region.

[0542] 45. The rodent or cell of clause 44, wherein the rodent comprises a complete repertoire of human V6 segments, a complete repertoire of human D6 segments, and a complete repertoire of human J6 segments at the rodent TCRa variable locus.

[0543] 46. The rodent or cell of clause 44 or 45, wherein the rodent or cell comprises a fully human V6 constant region at the at the rodent TCRa locus operably linked to human TCR variable region segments, and there is no rodent V6 constant region at the rodent TCRa variable locus, such that a fully human TCR6 is expressed.

[0544] 47. The rodent or cell of any preceding clause further comprising a repertoire of unrearranged human TCRy variable region segments, such as a one or more or a complete repertoire of human Vy segments, and one or more or a complete repertoire of human Jy segments, the variable region segments operably linked to a human TCRy constant gene sequence.

[0545] 48. A genetically modified rodent or rodent cell according to any preceding clause wherein the unrearranged human T cell variable region TCRa V and J gene segments are operably linked with the endogenous rodent TCRa enhancer downstream of TCRa constant region.

[0546] 49. A genetically modified rodent or rodent cell according to any preceding clause wherein the unrearranged human T cell variable region V, (D) and / or J gene segments of the TCRa and / or TCRP, and / or the TCRa and / or TCRP constant region, are operably linked to an endogenous rodent regulatory sequence or sequences, such as a rodent promoter or enhancer.

[0547] 50. A genetically modified rodent or rodent cell according to any preceding clause wherein the genome comprises (i) a nucleotide sequence encoding a human CD8a polypeptide which is operably linked to a rodent CD8a promoter and / or another rodent regulatory sequence or sequences and the nucleic acid sequence encoding a human CD8P polypeptide is operably linked to a rodent CD8P promoter and / or other rodent regulatory sequence or sequences, optionally where the rodent regulatory sequence is an endogenous rodent promoter or regulatory sequence; or

[0548] (ii) a nucleotide sequence encoding a chimaeric human-rodent CD8a polypeptide which is operably linked to a rodent CD8a promoter and / or another rodent regulatory sequence or sequences and the nucleic acid sequence encoding a chimaeric human-rodent CD8P polypeptide is operably linked to a rodent CD8P promoter and / or other rodent regulatory sequence or sequences, optionally where the rodent regulatory sequence is an endogenous rodent promoter or regulatory sequence.

[0549] 51. A genetically modified rodent or rodent cell according to any preceding clause wherein

[0550] (i) the genome comprises a nucleic acid encoding a human MHC class I polypeptide which is operably linked to a rodent MHC class I promoter, enhancer, and / or other rodent regulatory sequence, optionally where the rodent promoter or regulatory element(s) is an endogenous rodent promoter or regulatory sequence or

[0551] (ii) the genome comprises a nucleic acid sequence encoding a chimaeric MHC class I polypeptide which is operably linked to a rodent MHC class I promoter, enhancer, and / or other rodent regulatory sequence, optionally where the rodent promoter or regulatory element(s) is an endogenous rodent promoter or regulatory sequence.

[0552] 52. A genetically modified rodent or rodent cell according to any one of clauses 49- 51 wherein the endogenous rodent regulatory sequence is the naturally occurring rodent genomic regulatory sequence located at its natural genomic location.

[0553] 53. A genetically modified rodent or rodent cell according to any preceding clause wherein the human or chimaeric MHC class I polypeptide is selected from the group consisting of HLA-A, HLA-B, and HLA-C.

[0554] 54. A genetically modified rodent or rodent cell according to any preceding clause wherein the rodent genome comprises a rodent HLA locus comprising an insertion cassette, the cassette comprising site specific recombinase sites allowing insertion of human HLA DNA into the rodent genome by recombinase mediated cassette exchange, such that different human HLA molecules may be expressed from the same genomic location.

[0555] 55. A genetically modified rodent or rodent cell according to any preceding clause comprising DNA encoding human or chimaeric HLA-A02, and wherein the rodent H2D locus is inactive, optionally wherein the rodent H2D locus has been replaced by human or chimaeric DNA encoding human HLA- A02; further optionally wherein the rodent H2-K locus is deleted or inactivated.

[0556] 56. A genetically modified rodent or rodent cell according to any preceding clause comprising a nucleotide sequence encoding a human P2 microglobulin polypeptide at an endogenous non-human P2 microglobulin locus, wherein the rodent or rodent cell expresses the human P2 microglobulin polypeptide. 57. A genetically modified rodent according to any preceding clause wherein the nucleic acids encoding human TCRa and the human TCRP are in the germline of the rodent, and, where present, a human or chimaeric human-rodent CD8a polypeptide, a human or chimaeric human-rodent CD8P polypeptide, a human or chimaeric MHC class I polypeptide and / or human P2 microglobulin are in the germline of the rodent.

[0557] 58. A genetically modified rodent according to any preceding clause wherein

[0558] (i) the nucleic acids encoding a chimaeric human-rodent CD8a polypeptide, a chimaeric human-rodent CD8P polypeptide, a human TCRa, a human TCR p, a human MHC class I, and human P2 microglobulin, are comprised in the germline of the rodent; or

[0559] (i) the nucleic acids encoding a human CD8a polypeptide, a human CD8P polypeptide, a human TCRa, a human TCR p, a human MHC class I, and human P2 microglobulin are comprised in the germline of the rodent; or

[0560] (ii) the nucleic acids encoding a rodent CD8a polypeptide, a rodent CD8P polypeptide, a human TCRa, a human TCR p, a chimaeric human-rodent MHC class I, and human P2 microglobulin is comprised in the germline of the rodent.

[0561] 59. A genetically modified rodent or rodent cell according to any preceding clause wherein the rodent is a mouse or rat, preferably a mouse.

[0562] 60. A genetically modified rodent or rodent cell according to any preceding clause, comprising a TCRa variable gene locus comprising one or more, optionally all, of the following human Va gene segments:

[0563] TRAV1-1, TRAV1-2, TRAV2, TRAV3, TRAV4, TRAV5, TRAV6, TRAV8-1, TRAV8-2, TRAV8-3, TRAV8-4, TRAV8-6, TRAV9-1, TRAV9-2, TRAV10, TRAV12-1, TRAV12-2, TRAV12-3, TRAV13-1, TRAV13-2, TRAV14 / DV4, TRAV16, TRAV17, TRAV19, TRAV20, TRAV21, TRAV22, TRAV23 / DV6, TRAV24, TRAV25, TRAV26-1, TRAV26-2, TRAV27, TRAV29 / DV5, TRAV30, TRAV34, TRAV36 / DV7, TRAV38-1, TRAV38-2 / DV8, TRAV39, TRAV40, TRAV41 and / or comprising one or more, optionally all, of the following human Ja gene segments:

[0564] TRAJ3, TRAM, TRAJ5, TRAJ6, TRAJ7, TRAJ9, TRAJ10, TRAJ11, TRAJ12, TRAJ13, TRAJ14, TRAJ15, TRAJ16, TRAJ17, TRAJ18, TRAJ20, TRAJ21, TRAJ22, TRAJ23, TRAJ24, TRAJ26, TRAJ27, TRAJ28, TRAJ29, TRAJ30, TRAJ31, TRAJ32, TRAJ33, TRAJ34, TRAJ35, TRAJ36, TRAJ37, TRAJ38, TRAJ39, TRAJ40, TRAJ41, TRAJ42, TRAJ43, TRAJ44, TRAJ45, TRAJ46, TRAJ47, TRAJ48, TRAJ49, TRAJ50, TRAJ52, TRAJ53, TRAJ54, TRAJ56, TRAJ57.

[0565] 61. A genetically modified rodent or rodent cell according to any preceding clause, comprising a TCRP variable gene locus comprising one or more, optionally all, of the following human vp gene segments:

[0566] TRBV2, TRBV3-1, TRBV4-1, TRBV4-2, TRBV5-1, TRBV5-4, TRBV5-5, TRBV5-6, TRBV6-1, TRBV6-2, TRBV6-4, TRBV6-5, TRBV6-6, TRBV6-8, TRBV7-2, TRBV7-3, TRBV7-4, TRBV7-6, TRBV7-7, TRBV7-9, TRBV9, TRBV10- 1, TRBV10-2, TRBV10-3, TRBV11-1, TRBV11-2, TRBV11-3, TRBV12-3, TRBV12-4, TRBV12-5, TRBV13, TRBV14, TRBV15, TRBV16, TRBV18, TRBV19, TRBV20-1, TRBV24-1, TRBV25-1, TRBV27, TRBV28, TRBV29- 1, TRBV30 and / or comprising one or more, optionally all, of the following human jp gene segments:

[0567] TRBJ1-1, TRBJ1-2, TRBJ1-3, TRBJ1-4, TRBJ1-5, TRBJ1-6, TRBJ2-1, TRBJ2-2, TRBJ2-3, TRBJ2-4, TRBJ2-5, TRBJ2-6, TRBJ2-7.

[0568] 62. A genetically modified rodent or rodent cell according to any preceding clause, comprising a genome in which CD3e, CD3y, CD36 and / or CD3^ are endogenous wild type.

[0569] 63. A genetically modified rodent or rodent cell according to clause 62, wherein all of CD3e, CD3y, CD36 and CD3^ are endogenous wild type.

[0570] 64. A method for making a genetically modified rodent that expresses a human T cell receptor comprising:

[0571] (i) inserting into an endogenous rodent TCRa variable gene locus an unrearranged human TCRa variable gene locus comprising at least one human Va segment and at least one human Ja segment operably linked to a human TCRa constant region;

[0572] (ii) inserting into an endogenous rodent TCR variable gene locus an unrearranged human TCR variable gene locus comprising at least one human v segment, at least one human DP segment, and at least one human jp segment operably linked to a human TCRP constant region.

[0573] 65. A method according to clause 64 additionally comprising one or more or all of the following steps

[0574] (iii) inserting into an endogenous rodent CD8a gene locus a nucleic acid sequence encoding a human CD8a polypeptide;

[0575] (iv) inserting into an endogenous rodent CD8P gene locus a nucleic acid sequence encoding a human CD8P polypeptide;

[0576] (v) inserting into an endogenous MHC class I locus a nucleic acid sequence encoding a human MHC class I polypeptide; and

[0577] (vi) optionally inserting into an endogenous rodent P2 microglobulin gene locus a nucleic acid sequence encoding a human P2 microglobulin.

[0578] 66. A method according to clause 64 additionally comprising one or more or all of the following steps

[0579] (iii) inserting into an endogenous rodent CD8a gene locus a nucleic acid sequence encoding a chimaeric human-rodent human CD8a polypeptide, or modifying the host rodent CD8a gene locus by replacement of rodent DNA with human DNA to create DNA encoding chimaeric human-rodent human CD8a polypeptide; (iv) inserting into an endogenous rodent CD8P gene locus a nucleic acid sequence encoding a chimaeric human-rodent CD8P polypeptide, or modifying the host rodent CD8P gene locus by replacement of rodent DNA with human DNA to create DNA encoding a chimaeric human-rodent human CD8P polypeptide;

[0580] (v) inserting into an endogenous MHC class I locus a nucleic acid sequence encoding a human MHC class I polypeptide and

[0581] (vi) optionally inserting into an endogenous rodent P2 microglobulin gene locus a nucleic acid sequence encoding a human P2 microglobulin.

[0582] 67. A method according to clause 64 additionally comprising one or more or all of the following steps

[0583] (iii) inserting into an endogenous MHC class I locus a nucleic acid sequence encoding a chimaeric rodent-human MHC class I polypeptide; or modifying the host rodent MHC class I gene locus by replacement of rodent DNA with human DNA to create DNA encoding chimaeric human-rodent human MHC class I polypeptide;

[0584] (iv) optionally inserting an endogenous rodent P2 microglobulin gene locus with a nucleic acid sequence encoding a human P2 microglobulin

[0585] 68. A method according to any one of clauses 64-67 wherein the host loci at the site of each insertion are inactivated or deleted, such that the rodent does not express native rodent TCR, MHC class I or CD8, and optionally also not rodent P2 microglobulin.

[0586] 69 A method for making a fertile rodent or rodent cell, the method comprising the step of inverting the rodent TCRP in the rodent genome from at least rodent vpi to Dpi, optionally the method further comprising the insertion into a rodent genome of nucleic acid, such as DNA, encoding all or a part of a TCRP from another species which is different from the rodent, such as inserting human DNA encoding all or a part of TCRp.

[0587] 70. A method for making a genetically modified fertile rodent that expresses a human T cell receptor, the method comprising deletion of the rodent TCR p from at least rodent vpi to Dpi, including a deletion of serine proteases genes Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2, wherein one or more or all of the deleted serine proteases genes Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2 are re-inserted into the rodent genome.

[0588] 71. A method for making a fertile rodent comprising a genetic knock out of the endogenous T cell receptor beta chain polypeptide, the method comprising deletion of the rodent TCRP from at least rodent vpi to Dpi, including a deletion of serine proteases genes Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2, wherein one or more or all of the deleted serine proteases genes Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2 are re-inserted into the rodent genome.

[0589] 72 A method for making a fertile rodent, the method comprising, in either order: (i) deletion of the rodent TCRP genomic DNA from at least rodent vpi to Dpi, including a deletion of one or more or all of the serine proteases genes Prss58, Prss59, Prss3b,Try 4, Try5, Try 10, Prss3, Prssl, and Prss2, and

[0590] (ii) insertion of one or more or all of the serine proteases genes Prss58, Prss59, Prss3b,Try 4, Try5, Try 10, Prss3, Prssl, and Prss2 into the rodent genome.

[0591] 73 A method according to any one of clauses 69-72 wherein the method additionally comprises any one of the steps (i) - (vi) of clauses 59 and 60.

[0592] 74. A method according to any of clauses 69 to 73, wherein the deletion is homozygous.

[0593] 75. A rodent or rodent cell, or method for making a rodent or rodent cell according to any one of clauses 1-74, wherein the rodent genome comprises DNA encoding a fully human TCRP chain and comprising human V, D J and C gene segments, wherein the rodent or rodent cell is capable of expression of said fully human TCRP chain, and wherein the rodent genome comprises a human intergenic and / or a human intronic DNA sequence located between the human DI and human C2 gene segments, preferably being a human intergenic and / or intronic sequence that naturally occurs together with the human D and J gene segments, optionally wherein all of the human DNA sequence between the human DI and human C2 gene segments is genomic human DNA.

[0594] 76. A rodent or rodent cell having a genome comprising DNA encoding a fully human TCR beta chain and comprising human V, D, J and C gene segments, wherein the rodent or rodent cell is capable of expression of said fully human TCR beta chain, and wherein the rodent genome comprises a human intergenic and / or a human intronic DNA sequence located between the human DI and human C2 gene segments, preferably being a human intergenic and / or intronic sequence that naturally occurs together with the human D and J gene segments, optionally wherein all of the human DNA sequence between the human DI and human C2 gene segments is genomic human DNA.

[0595] 77. A rodent, rodent cell or method according to clause 75 or clause 76, wherein the TCRbeta chain comprises a sequence encoded by rearrangement of a V gene segment and segments from the DJC2 cluster in the locus.

[0596] 78. A method for making a human T cell receptor in a rodent, the method comprising inserting DNA encoding a fully human TCR beta chain and comprising human V, D, J and C gene segments into the rodent genome, wherein the rodent is capable of expression of said fully human TCR beta chain, wherein the insertion comprises a human intergenic and / or a human intronic DNA sequence located between the human DI and human C2 gene segments, preferably being a human intergenic and / or intronic sequence that naturally occurs together with the human D and J gene segments, optionally wherein all of the human DNA sequence between the human DI and human C2 gene segments is genomic human DNA. 79. A method according to clause 78 wherein the TCR comprises a TCRbeta variable region of a TCRbeta chain with a sequence encoded by rearrangement of a V gene segment and segments from the DJC2 cluster in the TCRbeta locus of the T cell.

[0597] 80. A method of producing a human T cell receptor to an antigen of interest comprising:

[0598] (i) optionally, making a rodent according to any preceding clause,

[0599] (ii) immunizing a rodent of any of the preceding clauses with the antigen of interest;

[0600] (iii) allowing the rodent to mount an immune response;

[0601] (iv) determining a nucleic acid sequence of the human TCR variable regions expressed by a T cell from the rodent reactive to the antigen of interest, optionally including a step of isolating the T cell and

[0602] (v) expressing the human T cell receptor, or human T cell receptor variable region in a cell; optionally further formulating the expressed human T cell receptor, or human T cell receptor variable region with a pharmaceutically acceptable excipient; or

[0603] (vi) inserting the nucleic acid encoding the human T cell receptor, or human T cell receptor variable region, into a cell, ex vivo or in vitro, such as into a human or animal cell, optionally wherein the cell containing the inserted nucleic acid is formulated for delivery to a human or animal, respectively; or

[0604] (vii) formulating a nucleic acid (e.g. RNA or DNA) encoding the human T cell receptor, or human T cell receptor variable region, with a suitably delivery vehicle, such as a lipid or liposome, for delivery to a patient in need thereof in vivo.

[0605] 81. A method according to clause 80 wherein the TCR comprises a TCRbeta variable region of a TCRbeta chain with a sequence encoded by rearrangement of a V gene segment and segments from the DJC2 cluster in the TCRbeta locus of the T cell.

[0606] 82. A method of treating an individual in need thereof, the method comprising

[0607] (i) delivery of a soluble TCR molecule comprising the human T cell receptor variable region of step

[0608] (v) of clause 80, and optionally comprising a human T cell receptor constant region, to the individual,

[0609] (ii) delivery of a cell according to step (vi) of clause 80 into the patient in need thereof,

[0610] (iii) delivery of a nucleic acid (e.g. RNA e.g. mRNA or DNA) encoding the human T cell receptor, or human T cell receptor variable region formulated as in step (vii) of clause 80 into a patient in need thereof. Examples

[0611] Example 1

[0612] Transgenic mice have been generated in which the mouse TRB VDJ locus was deleted, and also in which the TRB VDJ locus was inverted but retained in the genome.

[0613] The mouse variable region inversion is as shown in Figure 1, from coordinates 6:40868163 (GRCm39) to 6:41515110 (GRCm39).

[0614] The mouse constant region is deleted.

[0615] Homozygous male mice produced litters in 92% cases (n=12 male mice) when the mouse TRB VDJ locus was present in the natural or inverted orientation. However, in male mice where the mouse TRB VDJ locus was deleted, litters resulted in only 35% of cases (n=34 male mice). Therefore, the variable region inversion retains genetic components that, if deleted, significantly affect fertility.

[0616] Example 2

[0617] As noted herein, previously generated mice expressing human TRB DNA (see Moore et. al., Science Immunology, Humanization of T cell-mediated immunity in mice, 6(66) 2021) require the use of TRB murine D and J introns and intergenic sequences in the human DI - J2 region including the DJC2 gene cluster. Without re-murinisation T cell receptors from the DJC2 gene cluster were not seen. However, T cell receptors have been successfully generated from transgenic mice having human D and J introns at the mouse TRB DJC2 cluster without the need for murine TRB D and J introns or intergenic sequences.

[0618] The mouse comprises an inversion of the TCR beta variable locus, which significantly reduces, and may prevent, the production of TCR beta chains expressed from host mouse TCR beta chain variable gene segments, because the variable region is in an inverted position distal from the wild type locus.

[0619] Such mice comprise a fully human insertion of ~520KB of human genomic DNA from position 7:142,237,898 [GRCh38] to position 7:142,813,740 [GRCh38] in the mouse genome at position 6:36,074,375. As measured between the human genome coordinates, this represents an insertion of 576 kbp human DNA. The mice retain the murine TRB variable gene region in inverted form, as described above for example 1.

[0620] The mouse 3' enhancer downstream of the TrbC2 was retained.

[0621] Example 3

[0622] The Figures illustrate preferred examples of features of genome design. Each individual allele and the genomic intermediates described below are separate elements of the invention, combinable with other alleles and intermediates described herein.

[0623] Figure 1 illustrates the mouse TRA locus, the human TRA locus, and the engineered human mouse TRA locus. Human DNA is from chromosome 14:21,570,693 to 14:22,554,820 (GRCh38) in the third panel. Mouse DNA is numbered with respect to GRCm39. A preferred mouse locus contains human Tra (45V and 51J) and Trd (6V and 2J) DNA replacing the mouse Tra and Trd gene segments. Another preferred mouse locus contains human Tra (44V and 50J) and Trd (8V and 4J) DNA replacing the mouse Tra and Trd gene segments. The enhancer downstream of TraC is preferably retained.

[0624] A TRA locus of the invention was generated as follows: "'1.1 Mbp of KB (specifically 984,127 bp) of human genomic DNA from position 14:21,570,693 [GRCh38] to position 14:22,554,820 [GRCh38] was inserted into the mouse genome after mouse co-ordinate position: 14:52,664,870 or 14:52,664,818 (depending on the mouse line). The mouse TRA and Trd genetic loci were deleted from the mouse genome, from position 14:52,664,870 to 14:54,463,673, or from 14:52,664,818 to 14:54,463,673. The engineering of the locus comprises (i) a landing pad insertion along with large deletion of the mouse locus (ii) insertion of the human BACs using RMCE and (iii) deletion of selectable markers in the locus using piggyBac mediated excision, using techniques described in Lee et al., volume 32 number 4 April 2014, Nature Biotechnology, and Boroviak et al., Genesis 54:78-85 (2016)

[0625] Figure 2 illustrates the mouse TRB locus, the human TRB locus, and the engineered human mouse TRB locus in which the human DNA is inserted and the mouse DNA inverted, to preserve male fertility.

[0626] Human DNA is from chromosome 7:142,237,898 to 7:142,813,740 (GRCh38) in the third panel. Mouse DNA is numbered with respect to GRCm39. A preferred mouse locus contains human Trb (47V and 13J). A preferred mouse retains the murine TRb locus in the genome in an inverted orientation.

[0627] A TRB locus of the invention was generated as follows: "'520KB of human genomic DNA from position 7:142,237,898 [GRCh38] to position 7:142,813,740 [GRCh38] was inserted into the mouse genome at position 6:36,074,375. As measured between the human genome coordinates, this represents an insertion of 576 kbp human DNA. The inserted human DNA is joined to mouse genomic DNA at mouse position 6:41,535,764 (GRCm39). The naturally occurring mouse TCRbeta region from 6:40,868,163 (GRCm39) to 6:41,515,110 (GRCm39) was inverted in the genome using a lox mediated inversion process, using principles as described in Figure 1 b of Lee et al 2014 Nat Biotechnol 2014 Apr;32(4):356- 63, doi: 10.1038 / nbt.2825. Epub 2014 Mar 16. The mouse enhancer naturally located downstream of mouse trbc2 was present in the mouse genome downstream of human trbc2.

[0628] Figure 3a / b illustrates DNA encoding human CD8a replacing DNA encoding mouse CD8a in the mouse genome and illustrates DNA encoding human CD8P replacing mouse CD8P in in the mouse genome. A chimaeric CD8a and chimaeric CD8P polypeptide is generated. The location of the enhancers and DNase sites shown in Figure 3a is described in "Chromatin and CD4, CD8A and CD8B gene expression during thymic differentiation. Kioussis D, Ellmeier W. Nat Rev Immunol. 2002 Dec;2(12):909-19. doi: 10.1038 / nri952."

[0629] Figure 3b shows a process to make the genome of figure 3a in more detail using a dual targeting vector approach well known in the art, to generate chimaeric CD8a and CD8P loci, having 5' human DNA and 3' mouse DNA.

[0630] The two loci are in close linkage on mouse chromosome 6 and thus optimally they are humanised sequentially. Alternatively, it is in principle possible to modify them together, although given their distance from one another, targeting both together is technically more challenging.

[0631] To modify CD8b and CD8a sequentially either gene can be used as a starting point, the example Figure 3b shows CD8b first followed by CD8a, but it should be understood that CD8a could be humanized first followed by CD8b. A short segment of mouse CD8b from exon 1 to exon 3, along with the encoded intron sequence, is replaced with the equivalent region of human CD8B. The 1sttargeting vector contains homology arms flanking the region to be replaced, each around 3-4 kb in length. To provide positive selection the vector contains a puro-delta-tk selection marker flanked by PiggyBac inverted repeats. This selection marker is inserted into intron 2 of the human DNA insert. The exact position is not consequential because it is seamlessly removed with PBase in a second step, so for example the marker could be inserted in intron 1 or 3. Following transfection and selection in puromycin, targeted clones are identified by junction PCR on the 5' and 3' sides. Subsequently the targeting the vector is removed without trace by expression of PBase and cells where this has been achieved are selected in FIAU.

[0632] Cells in which CD8b has been successfully humanized can then be used to humanise CD8a as shown in Figure 3b using a similar vector design. Targeted clones are selected in puromycin and cells in which the selection marker has been removed are selected in FIAU, following PBase expression. Mouse CD8a sequences from exon 1 to exon 3 are replaced with human CD8a sequences from exon 1 to exon 4. The resultant chimaeric locus has 6 exons as a result of a fusion of human CD8a exon 4 with mouse CD8a exon 3. The second targeting can occur in cis or trans with the first. Clones need to be targeted in cis due to the close linkage of the genes. Clones with cis targeting are identified using methods well known in the art such as pulse field gel electrophoresis and Southern blotting.

[0633] In an alternative method, both CD8a and CD8b could be targeted together to generate the chimaeric locus. A first targeting vector is generated comprising human CD8b exon 1 - exon 3, and CD8a exon 1 - exon 4, separated by a selectable marker [e.g., PGK neo]. This is inserted into the mouse genome using homologous recombination and selection. The mouse CD8b exons 1-6 and CD8a exons 1-3 are deleted. A second targeting vector with a second selection marker [e.g., PGK puro delta TK] inserts the desired mouse genetic components of the CD8b chimaeric locus downstream of the human CD8b. These include a part of mouse exon 3, and all of mouse exon 4, 5 and 6. The second targeting vector also replaces the human 5' UTR of exon 1 of human CD8a with the mouse 5' UTR.

[0634] Following either construction method the end result is identical (Figure 3a). As well as human coding sequences for parts of CD8a and CD8P, the human intervening sequences between exon 1-2 and 2-3 are inserted into the rodent genome as a part of the human genomic insertion and (additionally for CD8A) the human intervening sequences between exon 3-4 are inserted in the rodent genome, preferably with the co-ordinates shown. The rodent CD8A locus comprises human exons 1 (excluding the UTR), 2, 3 and at least a part of human exon 4, fused to a part of mouse exon 3, all of mouse exon 4 and all of mouse exon 5. The rodent CD8B locus comprises human exons 1 (excluding the UTR), 2, and at least a part of human exon 3.

[0635] Figure 4 illustrates a hypothetical scheme by which DNA encoding the human CD8a could be used to replace DNA encoding mouse CD8a in the mouse genome and illustrates DNA encoding the human CD8P replacing mouse CD8P in in the mouse genome. A fully human CD8 polypeptide would be generated. The location of the enhancers and DNase sites shown is described in "Chromatin and CD4, CD8A and CD8B gene expression during thymic differentiation. Kioussis D, Ellmeier W. Nat Rev Immunol. 2002 Dec;2(12):909-19. doi: 10.1038 / nri952."

[0636] Figure 5a / b illustrates DNA encoding the human P2 microglobulin replacing DNA encoding mouse P2 microglobulin in the mouse genome. Figure 5b shows a process to make the genome of figure 5a in more detail. A preferred rodent genome comprises human Exon 1, 2 and 3 and intervening human genomic sequences, preferably with the coordinates shown.

[0637] A P2 microglobulin locus of the invention was generated by construction of a targeting DNA vector encoding human P2 microglobulin exon 1, 2 and 3, and the genomic intervening sequences. Targetting of that vector into the mouse genome replaces the equivalent of mouse exons 1-3 with sequences under the control of a mouse enhancer 5' of Exon 1. The targeting deletes the mouse exons 1-3 thereby preventing expression of mouse P2 microglobulin.

[0638] Figure 6a / b illustrates the human HLA_A*02:01 MHC class I gene replacing mouse H2-D1 in the mouse genome. Figure 6b shows a process to make the genome of figure 6a in more detail. A preferred rodent genome comprises the coding region of human Exons 1 - 8 and intervening human genomic sequences between exons 1-2, 2-3, 3-4, 4-5, 5-6, 6-7 and 7-8, preferably with the coordinates shown.

[0639] A modified MHC class 1 locus of the invention was generated by construction of a targeting DNA vector encoding human exons 1-7, and part of exon 8, and the genomic intervening sequences. Following homologous recombination to replace the mouse H2-D1 DNA (preventing expression of the mouse MHC H2-D1 DNA), a second targeting vector was used to replace human exon 4 for mouse exon 4. All other intervening DNA sequences were human. Such an approach results in a human MHC class I with a mouse exon 4, under the control of a mouse enhancer upstream of human exon 1. The mouse genome may optionally additionally have a knockout of, or mutation in, the H2K gene to prevent expression of host MHC class I molecules.

[0640] Whilst the Figures indicate certain preferred co-ordinates for the genome modification in mice, these are not limiting on the invention.

[0641] Example 4

[0642] Transgenic mice were created with a human TRA locus as shown in Figure 1 and / or a human TRB locus as shown in Figure 2. The hTCR transgenic mice generated by the present inventors may be referred to herein as OpTiMus® mice.

[0643] Table 1 shows the human TCR variable region gene segments theoretically present at the TRAV and

[0644] TRBV loci in the mice, based on the genes believed to be present in the BAC used to create the mice.

[0645] TRAV TRAJ TRBV TRBJ

[0646] 28TRAJ33 59TRAJ58 (ORF) 28TRBV12-3 58TRBV22-1 (P)

[0647] 29TRAJ34 60TRAJ59 (ORF) 29TRBV12-4 59TRBV23-1 (ORF)

[0648] 30TRAJ35 61TRAJ60 (P) 30TRBV12-5 60TRBV26 (P) 31TRAJ36 62TRAJ61 (ORF)

[0649] Table 1. Variable and joining region complement of OpTiMus® hTCR transgenic mice. TRAV genes: 44 functional and 10 non-functional genes. TRAJ genes: 50 functional and 12 non-functional genes. TRBV genes: 43 functional and 17 non-functional genes. TRBJ genes: 13 functional and 1 non-functional genes. Non-functional genes are annotated (P) Pseudogene and (ORF) Open Reading Frame.

[0650] The following genes were absent from human BAC sequences used in generation of the hTCR transgenic mice, so their expression is not possible:

[0651] TRBV4-3 (related to TRBV4-2, 4-1 and TRBV1)

[0652] TRBV5-8 (related to TRBV5-5,5-3,5-7,5-4 etc.) TRBV6-3 (identical to TRBV6-2)

[0653] TRBV6-9 (related to TRBV6-5,6-6,6-8,6-7 etc.)

[0654] TRBV7-8 (related to TRBV7-4, 7-6, 7-7, etc.) Expression of gene segments was assessed by sequencing bulk RNA extracted from OpTiMus® mouse spleen. All transcripts that could be assigned to an individual gene segment were counted, whether expressed in-frame or out-of-frame. To reduce PCR bias, we counted UM I (unique molecular identifiers) rather than the number of sequence reads. Two transcripts (different UMIs) may have the same nucleotide sequence. Detection of at least one transcript from a gene segment confirms that the gene segment is present and capable of being expressed.

[0655] A) VJ segment use in TRBV

[0656] Within the transgenic human BAC sequences there are 43 functional TRBV genes and 13 functional TRBJ genes (Table 1). Bulk spleen RNA sequencing of TRB genes expressed in homozygous TRB transgenic mice demonstrated broad recombination and expression of TRBV and TRBJ genes (Figure 10, Figure 11, Figure 12). As each TRBD is associated with a TRBJ and constant region, it is expected that the full complement of TRBD gene segments is also capable of being expressed.

[0657] All of the functional TRBJ and TRBV genes were represented in the bulk sequencing results. This confirms that all functional human gene segments of the TCRP variable region are expressed from the transgenic mouse locus.

[0658] B) VJ segment use in TRAV

[0659] Within the transgenic human BAC sequences there are 44 functional TRAV genes and 50 functional TRAJ genes (Table 1). Bulk spleen RNA sequencing of TRB genes expressed homozygous TRA transgenic mice demonstrated broad recombination and expression of TRAV and TRAJ genes.

[0660] All of the functional TRAJ genes and all but two of the functional TRAV genes (TRAV7 and TRAV18), were represented in the bulk sequencing results (Figure 13, Figure 14, Figure 15). This confirms expression of the TCRa human gene segments from the transgenic mouse locus.

[0661] C) Relative usage of TRBJ1-C1 and TRBJ2-C2

[0662] Bulk sequencing of TRB transgenic spleen RNA indicate an approximate 40:60 split for use of TRBJ1- C1:TRBJ2-C2 (Figure 16). D) Comparison with TCR gene segment expression in humans

[0663] The repertoire of peripheral TCR expression can be envisaged as being shaped by two processes: (i) the propensity for certain VDJs to undergo somatic recombination and (ii) the thymic positive and negative selection that is dependent upon the pMHC that the TCR encounters during thymic development.

[0664] Two published datasets of TCR gene expression in healthy humans were compared to the results from OpTiMus® mice. Freeman et al (Genome Res 19(10):1817-1824, 2009) reported TRBV / TRBJ expression in PBMC RNA pooled from 550 healthy individuals. Kitaura et al (BMC Immunologyl7:38, 2016) reported TRAV / TRAJ and TRBV / TRBJ expression from 20 healthy Japanese individuals.

[0665] TRBV and TRBJ gene usage in Optimus® mice was compared with published data from humans. TRB transgenic mice and humans express broadly similar TRBV and TRBJ genes. Notably, TRBV20.1 and TRBV5.1 are the two most highly expressed genes in humans and in the transgenic mice (Figure 11, Figure 17). There is also a very close alignment between TRBJ usage in humans and in the transgenic mice (Figure 18).

[0666] The length of CDR3 region, which spans the highly variable VDJ joining region, ranged from about 20 bp to 60 bp, with a peak at 42 bp, which is indistinguishable from that found in humans (Kitaura et al, 2016) - Figure 19.

[0667] The approximate 40:60 TRBC usage split in favour of TRBJ2-C2 seen in humans is also recapitulated in the transgenic mice (Figure 16).

[0668] TRAV and TRAJ gene usage in OpTiMus® mice was compared with published data from humans. Most of the TRAVs that express well in the human data set also express in the mice. Compared with the human data set, there is relatively little expression at the distal end of the TRAV locus (Figure 14, Figure 20). Good expression levels are seen throughout functional TRAJ genes in humans and in TRA transgenic mice (Figure 15, Figure 21).

[0669] The length of CDR3 region, which spans the highly variable VJ joining region, ranged from about 24 bp to 57 bp, with a peak at 42 bp, which is indistinguishable from that found in humans (Kitaura et al, 2016) - Figure 19.

[0670] Furthermore, the junctional diversity created by recombination of v and j gene segments (Figure 10, Figure 13) also reflects the junctional diversity seen in humans. This is significant because the junction between the gene segments forms the CDR3 of the TCR - the most variable component and that which directly interacts with the peptide. E) Comparison between OpTiMus® mice and other humanized TCR mice

[0671] Humanized TCRab mice have been described by two companies - Regeneron with their VelociT mice and T-knife with their MyT mice.

[0672] The Regeneron humanized mouse ("VelociT") contains humanized gene targeted TRA / TRB / CD8ab / CD4 / b2m / MHC I and MHC II (Moore etal, Sci. Immunol. 6, eabj4026 2021). The TRA and TRB contain murine constant region genes. The initial version of the Regeneron mouse that had fully human TRBJ1 and TRBJ2 sequences only expressed the Jl-Cl cluster; conversion of the intergenic JC regions to mouse corrected the defect. The OpTiMus® mouse did not encounter this issue and expressed both TRBJ clusters in similar proportions to those found in humans.

[0673] The T-Knife mice (Li et al, Nature Medicine 16:9. 1029-1034 2010) were created by randomly integrating TRA and TRB containing YAC clones on a Tra / Trb knockout background and also contain humanized MHC Class I fused to b2-microglobulin. Assessment of TCR usage and expression was indicated by the simple presence or absence of PCR products amplified, either from recombined VJ genes or by RT PCR from RNA, without quantitative information on the relative expression of the different genes.

[0674] The expression status does not appear to have been reported for every V and J gene in the genomic regions inserted in the prior transgenic mice. Figure 22 summarises the expression status of the genes in the TCR loci of the prior transgenic mice from published data (Moore et al., 2021 and Li et al, 2010 cited above), compared against the expression status of genes in the TCR loci of the OpTiMus mice characterised herein.

[0675] The bulk sequencing analysis of OpTiMus® mice revealed that expression of inserted TCR gene segments was complete or almost complete. The only transcripts that were not detected in this analysis, wereTRAV7 and TRAV18. Notably present is the TRBJ2-6 gene (Figure 10, Figure 12, Figure 22d), whose absence in the Regeneron mouse was ascribed as being due to Trbj2-6 in mice being a pseudogene. In OpTiMus® mice, TRBJ2-6 was clearly expressed and at a similar level to that seen in humans.

[0676] Data for confirmed expression of gene segments in the transgenic mice are shown in Figure 22 and summarised in Table 2 below. OpTiMus Regeneron T-knife

[0677] TRAV 95% 77% 86%

[0678] TRAJ 100% 94% 76%

[0679] TRBV 100% 84% 65%

[0680] TRBJ 100% 92% 69%

[0681] Table 2. Confirmed gene expression in hTCR mice

[0682] Based upon the current analysis of gene expression in the OpTiMus® TRA and TRB mouse lines, it appears that an OpTiMus® mouse with transgenic TCRaP loci would have the most diverse human repertoire yet seen in any humanized TCR mouse.

[0683] Example 5

[0684] OpTiMus® mice were produced with the following genotypes, being homozygous ("horn"), heterozygous ("het") or wild type ("WT") at the loci indicated.

[0685] Table 3. Genotypes of OpTiMus mice used to assess naive immune cell populations. "Zygosity code" is a shorthand for the mouse genotype with reference to the transgenic status and zygosity of each of the loci containing insertion of the human transgenes shown in the table, e.g., a mouse homozygous for the transgene HLA-A*02, homozygous for human B2m, heterozygous for human CD8a and CD8b, homozygous for human TRA and heterozygous for human TRB is referred to as zygosity code E. A heterozygous locus has one human transgenic allele and one wild type (endogenous mouse) allele; a homozygous locus has two human transgenic alleles; a wild type (WT) locus has two endogenous alleles, with no human transgene insertion at the locus.

[0686] Loci were as follows:

[0687] H2K inactivated mouse MHC class I locus H2K HLA-A*02 mouse MHC I locus H2D containing insertion of nucleic acid encoding fully human HLA- A*02, replacing mouse gene, as shown in Figure 6a hB2M mouse P2-microglobulin locus containing insertion of nucleic acid encoding fully human

[0688] P2m, replacing mouse gene, as shown in Figure 5a hCD8a / b mouse CD8 locus containing insertion of nucleic acid encoding fully human CD8a and fully human CD8b, replacing corresponding mouse genes, as shown in Figure 3a hTRA mouse TCRa locus containing insertion of nucleic acid encoding fully human TRAV and

[0689] TRAC, replacing corresponding mouse genes, as shown in Figure lb hTRB mouse TCRP locus containing insertion of nucleic acid encoding fully human TRBV and

[0690] TRBC, with inactivating inversion of endogenous mouse TRBV region and deletion of mouse TRBC, as shown in Figure 2b.

[0691] No other transgenes were introduced, so all other genes were believed to be WT.

[0692] T lymphocyte populations and cell surface expression of CD8 were assessed in blood samples from antigen- naive OpTiMus® mice 1 to 7. Fresh blood was assessed using FACS to detect mouse CD3e and human CD8b. Results are shown in Figure 23. CD3+ cells were detected in all mice. Regular levels of hCD8+ cells, in the range of 1 - 5 % of total blood PBMC, were detected in all mice other than mouse 2, which was expected because mouse 2 expressed only mouse CD8 which would not be detected using the probe for human CD8b. Levels of CD3+ cells and CD8+ cells were within a normal range, comparable to those of wild type mice, indicating that T lymphocytes in the transgenic mice develop normally.

[0693] Further, immune cells were collected from spleens of three additional antigen naive fully homozygous mice (mouse 8, 9 and 10, having the same fully homozygous genotype as mouse 7 in Table 3 above) and stained for cell surface markers mCD3, mCD4, hCD8b, hTRBC-1 and mCD19 (Figure 24). Naive mice with fully humanised, fully homozygous transgenes (HLA, B2M, CD8a, CD8b, TRA and TRB), indicated as zygosity code K in Table 3, expressed a normal range of lymphocyte populations, with B cells 57-65%, CD4+ T cells 10-15% and CD8+ T cells 2.6-4.5%. In addition, CD3+ T cell populations showed TRBC-1 subpopulation of 5-7.5% and TRBC-1 negative subpopulation of 4.1-5.8%. The data demonstrate that the mice exhibit normal production of key lymphocytes populations, including CD4 and CD8 T cells and B cells.

[0694] Gene expression libraries were created from splenocytes of mouse 8, 9 and 10 using single cell mRNA sequencing technology in addition to cell surface marker staining. Immune cell types and T cell subset phenotypes were classified based on gene expression profile including B cells, T cells, NK cells, monocytes, dendritic cells (eDC) and others. The proportions of total splenocytes identified as CD4 T cell, CD8 T cell and gamma / delta T cells were within the range commonly known for murine splenocytes. CD4 T cells represented 11-14% of whole splenocyte population, whereas CD8 T cell represented 5-9.5% in the splenocytes. Gamma delta T cells represented around 1% of the whole population. Figure 25.

[0695] Example 6

[0696] T cell immune responses to peptide immunisation were characterised in OpTiMus® mice with genotypes of various zygosities.

[0697] Mice were immunised with purified target peptide in combination with adjuvants (incomplete Freund's adjuvant and CpG ODN 1826). The target peptide in this study is a cancer-associated T cell epitope, known to be presented on HLA-A*02.

[0698] Material for Tissue preparation / Cell staining and sorting

[0699] Medium: Tissue prep / culture media: RPMI-1640+10% FBS+2mM L-glutamine + 20mM HEPES; cell enrichment / staining buffer: PBS+2% FBS+20mM HEPES + ImM EDTA; Sorted cell collection buffer: PBS+20%FBS+20mM HEPES. Cell freezing Media: FBS+10%DMSO.

[0700] Reagents: EasySep Mouse CD8+ T cell isolation kit (STEMCELL, #19853), Anti-human CD8b antibody-FITC (Miltenyi, 130-11-567), anti-human CD8b-APC (Biolegend, 376705), anti-human CD8b-Viablue (Miltenyi, 130-110-515), anti-mouse CD8b-FITC (Biolegend, 126605), anti-mouse CD3e-FITC (biolegend 100306), anti-mouse CD3-BV421, (biolegend, 100228), anti-mouse TCRbeta-BV421 (Biolegend, 109230), antihuman TRBC-l-FITC (Biolegend, 383510), anti-mouse CD4-PE (Biolegend, 130310), anti-mouse CD19- BV605 (biolegend, 115539), eFluor 780 Fixable Viability Dye (ThermoFisher, 65-0865-18), Lightening link barcode conjugation kit (Abeam, ab270703 / 05 / 09), TotalSeq-C series-hashtag antibodies (Biolegend, 155861, 155863, etc. ).

[0701] Tetramers for detection of antigen specific CD8 T cells, obtained from MBL.

[0702] Methods

[0703] Immunisation method: Peptide based Immunisation

[0704] Immunogen: Purified peptides (>95% purity, Genscript)

[0705] Adjuvants: Incomplete Freund's adjuvant (IFA, Invivogen), CpG ODN 1826 (Invivogen)

[0706] Prime dose (per mouse): lOOpg of peptide and 50pg of CpG ODN / 50pl PBS emulsified with 50pl of IFA

[0707] Boosts dose (per mouse): 50pg of peptide and 50pg of CpG ODN / 50pl PBS emulsified with 50pl of IFA

[0708] Peptide based immunogen given via S.C. route of administration. Prime and two to three boosts schedule were typically used for these studies with 21 days gap between prime and boost and 14 days gap between each boost. For alternative prime boost regimen, weekly boosts were also used. Tissues were typically collected day 7 post each boost.

[0709] Tissue preparation, CD8+ T cell enrichment and staining for sorting

[0710] Mesenteric lymph nodes, inguinal lymph nodes and whole spleen were collected from each mouse for sorting. Briefly, lymph nodes and spleen are diced and pushed through a 40pM cell strainer to generate an even cell suspension. Cells were further enriched to CD8+ T cell suspension (typically 80% pure) by using Stemcell kit (19853). Enriched cells were spun down and kept on ice for staining.

[0711] MBL tetramers were used at 0.05-0. lpg / lOOpI final incubation volume for both target tetramer-PE and negative control tetramer-APC per stain for up to 2.5M enriched CD8+ murine T cells. After the addition of tetramers, cells were kept on ice and incubated for 15 mins. This was followed by adding a staining cocktail containing anti-hCD8b-viablue (Miltenyi), anti-hTRBC-l-FITC (Biolegend) and eFluor 780 Fixable Viability Dye. TotalSeq-C series-hashtag antibodies were also added into individual mouse samples for hash tagging purpose at this point. Cells were incubated for a further 30 mins on ice before washing with staining buffer twice.

[0712] Tetramer specific CD8+ T Cell sorting

[0713] Cells were resuspended in 300-400 pl of Staining buffer and pushed through a 35 pM cell strainer and kept on ice before acquiring on a cell sorter.

[0714] Cells were gated using the following gating strategy: Lymphocyte > Single cell > Live > hCD8b+ > Target tetramer+ & negative control tetramer-. Cells were sorted into a 1.5 ml microtube with ~300 pl of cold collection buffer.

[0715] Immune response rate was defined based on the % CD8+ lymphocyte population that were specific for the target antigen, measured by antigen specific tetramer gating in FACS, and comparing against control (non-immunised) mice. Mice exhibiting an immune response have a higher % antigen specific cell population compared with control mice. The % in responders was typically at least >0.1% of the population, whereas control % was typically <0.05% of the population.

[0716] Single cell mRNA-Seq , Library preparation and NGS

[0717] Freshly collected target cells were immediately taken for mRNA extraction for single cell RNA- sequencing and a mouse TCR VDJ library, Gene Expression (GEX) library and cell surface protein library were generated. Final libraries were quality controlled before being sequenced. Single cell NGS data were analysed to determine whether responding TCR clonotypes had fully human TCRs, focusing on CD8+ cells exhibiting clonal expansion and showing activated / exhausted- like / proliferating phenotypes.

[0718] Results In the first study, hTCR mice with alleles in various zygosity received peptide-based immunisation, and splenocytes were stained for antigen specific tetramer binding and sorted to collect target tetramer specific CD8+ populations.

[0719] Table 4. Summary of antigen specific CD8+ immune responses in OpTiMus mice of various zygosities.

[0720] Loci are as described in Example 5.

[0721] Table 5. OpTiMus mouse zygosity and immunological status for mice with data shown in Figures 23-28.

[0722] Figure 26a shows representative plots from 6 mice with different zygosity (zygosity B, D, E, I, J, K - Table 4) which have generated responses from cells with fully humanised TCR. Plot from Mouse 16 shows the level of target tetramer is 0.015% of total CD8+ population for a naive control mouse. Immunised mice with 6 different zygosity showed a range of tetramer specific population of 0.18% - 1.74%. This indicates that not only fully humanised homozygous mice (zygosity K) but also other zygosity can be used for discovery of full human TCRs.

[0723] Two mice (mouse 2 and mouse 28) with murine WT CD8 (zygosity G) did not respond to the same type of immunisation as those with human CD8. Figure 26b. This indicates that human CD8 enables signalling competency of human TCRa / p TCRs in this immunisation context with homozygous fully human MHC class I.

[0724] T cell responses for mice heterozygous for TRB and homozygous for TRA (zygosity I) versus homozygous for both TRB and TRA (zygosity K) were specifically examined.

[0725] In Figure 27 , OptiMus mice with zygosity I show antigen specific CD8+ T cell responses to weekly boost peptide-based immunisation. A total of 10 OptiMus mice were immunised with target peptide using the weekly boost regimen. Data were collected from 2 mice, 2 mice, 3 mice and 3 mice for boostl, boost2, boost3 and boost4 respectively. Mice numbered 17, 18, 19, 20 are representative of data for boost 1, 2, 3, 4 respectively. The data show that OptiMus mice homozygous for TRA and heterozygous for TRB (zygosity I) can generate antigen specific T cell responses from a prime-boost regimen with a single or multiple boost injections. Moreover, they generated a wide range of responses (from 0.8% of the whole CD8 population to 4.5%) from separate individuals.

[0726] In Figure 28, OptiMus mice with fully humanised alleles (zygosity K) show antigen specific CD8+ T cell responses with peptide-based immunisation. A total 23 OptiMus mice were immunised with target peptide using the biweekly boost regimen. Data were collected 7 days post boost2 and boost3. Mice numbered 16 and 24 are naive controls for boost2 and boost3 respectively. Mice 21, 22 and 23 are representative of data for boost2 (Figure 28a) , while mice numbered 25, 26, 27 are representative of data for boosts (Figure 28b). OptiMus mice with zygosity K show a wide range of responses from individuals and marked antigen specific responses from both boost2 (0.2~l%) and boosts (0.1~4.7%). Moreover, overall response rate of those 23 mice were 21 out 23 (>90%) indicating a high response rate.

[0727] Comparing the immune responses from homozygotes and heterozygotes for the modified alleles, it is notable that the strongest hTCR immune responses were achieved in animals lacking mouse TCRa. Although mice that were heterozygous at the TRA locus were still able to generate fully human TCRs in response to immunisation (e.g., mouse 11), animals with homozygous human TCRa were generally a better source of human TCRs than those that were heterozygous at the TCRa locus. This may be the result of the endogenous mouse TCRa constant domain out-competing the human TCRa constant domain for binding to CD3 in heterozygotes, generating predominantly T cells with mouse TCRa, whereas in homozygotes this cannot occur since mouse TRAC is not expressed. Immunisation of mice homozygous at the TCRa locus (TRA), and either homozygous or heterozygous at the TCRP locus (TRB), generated target-specific CD8+ T cell responses, confirming the utility of these mice for discovery of fully human TCR sequences with therapeutic potential.

[0728] Example 7

[0729] Matings were set up with 17 male mice having homozygous insertion of human TRB at the endogenous TRB locus, with endogenous TRBV inverted to an ectopic location by inversion of a chromosomal region, as shown in Figure 2. Breeding data are shown in Table 6.

[0730] Table 6. Fecundity of male mice with homozygous hTRB insertion and mTRBV inversion

[0731] All matings generated litters, confirming the fertility of mice retaining mouse TRBV displaced from the endogenous locus. This avoids the undesirable loss of fertility that was observed in male mice in which TRBV was deleted (Example 1).

[0732] Example 8

[0733] 124 T cell receptor sequences, isolated as antigen-binders from mice immunised with target peptide as described in Example 6, were chosen for expression in soluble "T-ceptor" format. TCR alpha and beta variable domains from the mice were incorporated into bivalent soluble molecules comprising two TCR "Fab"-like regions, dimerised via an antibody Fc region. Each TCR antigen-binding "Fab" was composed of a fully human alpha and a fully human beta polypeptide chain: an alpha chain comprising the human TCR a variable domain from the mouse linked at its C terminus to a human TCR a constant domain, paired with a beta chain comprising the human TCR p variable domain from the mouse linked at its C terminus to a human TCR constant domain. TCRs included diverse combinations of va and vP gene segments (Table 7).

[0734] Table 7. TCR germline genes of selected antigen-specific clonotypes

[0735] Nucleic acids encoding soluble TCRs were transfected into Expi293T cells for small scale (3 ml) protein expression. Proteins were purified using Protein A affinity purification five days post transfection.

[0736] Concentrations of soluble TCRs were measured using UV / Vis spectrophotometry. All TCR molecules were expressed and concentrations of purified protein ranged from 0.01 - 1.9 mg / ml. (Figure 29).

Claims

Claims1. A genetically modified rodent, comprising:(i) an unrearranged T cell receptor (TCR) a variable gene locus comprising at least one human Va segment and at least one human Ja segment, wherein the TCRa variable gene is operably linked to a human TCRa constant gene sequence,(ii) an unrearranged TCRP variable gene locus comprising at least one human vp segment, at least one human DP segment, and at least one human jp segment, wherein the unrearranged TCRP variable gene is operably linked to a human TCRP constant gene sequence wherein the unrearranged human T cell variable region gene segments are capable of rearranging to form genes that encode human T cell receptor variable domains, wherein the unrearranged TCRa variable gene locus is present at an endogenous rodent TCRa gene locus and the unrearranged TCRP variable gene locus is present at an endogenous rodent TCRP gene locus.

2. A genetically modified rodent according to claim 1, comprising:(i) an unrearranged T cell receptor (TCR) a variable gene locus comprising at least one human Va segment and at least one human Ja segment, wherein the TCRa variable gene is operably linked to a human TCRa constant gene sequence,(ii) an unrearranged TCRP variable gene locus comprising at least one human vp segment, at least one human DP segment, and at least one human jp segment, wherein the unrearranged TCRP variable gene is operably linked to a human TCRP constant gene sequence wherein the unrearranged human T cell variable region gene segments are capable of rearranging to form genes that encode human T cell receptor variable domains, wherein the unrearranged TCRa variable gene locus is present at an endogenous rodentTCRa gene locus and the unrearranged TCRP variable gene locus is present at an endogenous rodent TCRP gene locus and wherein the rodent genome comprises either[A] a nucleic acid sequence encoding a human CD8a polypeptide, wherein the nucleic acid sequence encoding a human CD8a polypeptide is present at an endogenous rodent CD8a co-receptor locus, a nucleic acid sequence encoding a human CD8P polypeptide, the nucleic acid sequence encoding a human CD8P polypeptide is present at an endogenous rodent CD8P co-receptor locus; and a nucleic acid sequence encoding a human MHC class I polypeptide present at an endogenous rodent MHC class I locus; or[B] a nucleic acid sequence encoding a chimaeric human-rodent CD8a polypeptide, wherein the nucleic acid sequence encoding a chimaeric human-rodent CD8a polypeptide is present at an endogenous rodent CD8a co-receptor locus, a nucleic acid sequence encoding a chimaeric human-rodent CD8P polypeptide, the nucleic acid sequence encoding a chimaeric human-rodent CD8P polypeptide is present at an endogenous rodent CD8P co-receptor locus, and a nucleic acid sequence encoding a human MHC class I polypeptide present at an endogenous rodent MHC class I locus; wherein the chimaeric CD8 protein is capable of binding to the human MHC class I; or[C] a nucleic acid sequence encoding a rodent CD8a polypeptide at the endogenous locus and a nucleic acid sequence encoding a rodent CD8 p polypeptide at the endogenous locus; and a nucleic acid[_sequence encoding a chimaeric human-rodent MHC class I polypeptide present at an endogenous rodent MHC class I locus, wherein the rodent CD8 protein is capable of binding to the chimaeric MHC class I, optionally wherein then rodent genome comprises a nucleic acid sequence encoding a human P2 microglobulin polypeptide which, when present, is located at an endogenous rodent P2 microglobulin locus.

3. A genetically modified rodent cell, such as an ES cell, comprising:(i) an unrearranged T cell receptor (TCR) a variable gene locus comprising at least one human Va segment and at least one human Ja segment, wherein the TCRa variable gene is operably linked to a human TCRa constant gene sequence,(ii) an unrearranged TCRP variable gene locus comprising at least one human vp segment, at least one human DP segment, and at least one human jp segment, wherein the unrearranged TCRP variable gene is operably linked to a human TCRP constant gene sequence wherein the unrearranged human T cell variable region gene segments are capable of rearranging to form genes that encode human T cell receptor variable domains, wherein the unrearranged TCRa variable gene locus is present at an endogenous rodent TCRa gene locus and the unrearranged TCRP variable gene locus is present at an endogenous rodent TCRP gene locus.

4. A genetically modified rodent cell according to claim 3, comprising:(i) an unrearranged T cell receptor (TCR) a variable gene locus comprising at least one human Va segment and at least one human Ja segment, wherein the TCRa variable gene is operably linked to a human TCRa constant gene sequence,(ii) an unrearranged TCRP variable gene locus comprising at least one human vp segment, at least one human DP segment, and at least one human jp segment, wherein the unrearranged TCRP variable gene is operably linked to a human TCRP constant gene sequence wherein the unrearranged human T cell variable region gene segments are capable of rearranging to form genes that encode human T cell receptor variable domains, wherein the unrearranged TCRa variable gene locus is present at an endogenous rodent TCRa gene locus and the unrearranged TCRP variable gene locus is present at an endogenous rodent TCRP gene locus and wherein the rodent genome comprises either[A] a nucleic acid sequence encoding a human CD8a polypeptide, wherein the nucleic acid sequence encoding a human CD8a polypeptide is present at an endogenous rodent CD8a co-receptor locus, a nucleic acid sequence encoding a human CD8P polypeptide, the nucleic acid sequence encoding a human CD8P polypeptide is present at an endogenous rodent CD8P co-receptor locus; and a nucleic acid sequence encoding a human MHC class I polypeptide present at an endogenous rodent MHC class I locus; or[B] a nucleic acid sequence encoding a chimaeric human-rodent CD8a polypeptide, wherein the nucleic acid sequence encoding a chimaeric human-rodent CD8a polypeptide is present at an endogenous rodent CD8a co-receptor locus, a nucleic acid sequence encoding a chimaeric human-rodent CD8P polypeptide, the nucleic acid sequence encoding a chimaeric human-rodent CD8P polypeptide is present at an endogenous rodent CD8P co-receptor locus, and a nucleic acid sequence encoding a human MHCclass I polypeptide present at an endogenous rodent MHC class I locus; wherein the chimaeric CD8 protein is capable of binding to the human MHC class I; or[C] a nucleic acid sequence encoding a rodent CD8a polypeptide at the endogenous locus and a nucleic acid sequence encoding a rodent CD8 p polypeptide at the endogenous locus; and a nucleic acid sequence encoding a chimaeric human-rodent MHC class I polypeptide present at an endogenous rodent MHC class I locus, wherein the rodent CD8 protein is capable of binding to the chimaeric MHC class I, optionally wherein then rodent genome comprises a nucleic acid sequence encoding a human 2 microglobulin polypeptide which, when present, is located at an endogenous rodent 2 microglobulin locus.

5. A genetically modified rodent T cell, comprising:(i) an unrearranged T cell receptor (TCR) a variable gene locus comprising at least one human Va segment and at least one human Ja segment, wherein the TCRa variable gene is operably linked to a human TCRa constant gene sequence,(ii) an unrearranged TCR variable gene locus comprising at least one human vp segment, at least one human DP segment, and at least one human jp segment, wherein the unrearranged TCRP variable gene is operably linked to a human TCRP constant gene sequence wherein the unrearranged human T cell variable region gene segments are capable of rearranging to form genes that encode human T cell receptor variable domains, wherein the unrearranged TCRa variable gene locus is present at an endogenous rodent TCRa gene locus and the unrearranged TCRP variable gene locus is present at an endogenous rodent TCRP gene locus.

6. A genetically modified rodent or rodent cell according to any one of claims 1-5, which does not express a functional endogenous TCRa polypeptide from the endogenous TCRa variable gene locus, and which is homozygous at the TCRa gene locus, and which does not express a functional endogenous TCRP polypeptide from the endogenous TCRP variable gene locus, and which is homozygous at the TCRP gene locus.

7. A genetically modified rodent or rodent cell which is either(i) a rodent or rodent cell according to any preceding claim, wherein the rodent retains an endogenous TCRa variable gene locus and / or an endogenous rodent TCRP variable gene locus, and wherein any retained endogenous rodent TCRa variable gene locus is a non-functional locus and / or wherein any retained endogenous rodent TCRP variable gene locus is a non-functional locus, optionally wherein all or a part of the retained endogenous rodent TCRa and / or p variable gene locus is inverted with respect to its normal orientation in the rodent genome; or(ii) A fertile rodent or rodent cell wherein the rodent TCRP locus from at least rodent vpi to Dpi is inverted within the rodent genome with respect to the natural orientation in the rodent genome, optionally the method further comprising the insertion into a rodent genome of nucleic acid, such as DNA,encoding all or a part of a TCRP from another species which is different from the rodent, such as inserting human DNA encoding all or a part of TCRp.

8. A genetically modified male rodent which is either(i) a rodent according to any preceding claim having substantially wild type fertility, wherein the rodent TCRP locus is deleted from at least rodent vpi to Dpi, and the genome additionally comprises one or more or all of the deleted serine proteases genes Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2(ii) a rodent having substantially wild type fertility, wherein the rodent TCRP locus has been deleted from at least rodent vpi to Dpi, and the genome additionally comprises one or more or all of the deleted rodent serine proteases genes Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2, wherein the rodent genome is not a wild type rodent genome, and / or does not have a wild type genome at the TCRP locus, and / or cannot make a rodent TCRP chain.

9. A genetically modified rodent or rodent cell according to any preceding claim, comprising a TCRa variable gene locus comprising one or more, optionally all, of the following human Va gene segments:TRAV1-1, TRAV1-2, TRAV2, TRAV3, TRAV4, TRAV5, TRAV6, TRAV8-1, TRAV8-2, TRAV8-3, TRAV8-4, TRAV8- 6, TRAV9-1, TRAV9-2, TRAV10, TRAV12-1, TRAV12-2, TRAV12-3, TRAV13-1, TRAV13-2, TRAV14 / DV4, TRAV16, TRAV17, TRAV19, TRAV20, TRAV21, TRAV22, TRAV23 / DV6, TRAV24, TRAV25, TRAV26-1, TRAV26- 2, TRAV27, TRAV29 / DV5, TRAV30, TRAV34, TRAV36 / DV7, TRAV38-1, TRAV38-2 / DV8, TRAV39, TRAV40, TRAV41 and / or comprising one or more, optionally all, of the following human Ja gene segments:TRAJ3, TRAJ4, TRAJ5, TRAJ6, TRAJ7, TRAJ9, TRAJ10, TRAJ11, TRAJ12, TRAJ13, TRAJ14, TRAJ15, TRAJ16,TRAJ17, TRAJ18, TRAJ20, TRAJ21, TRAJ22, TRAJ23, TRAJ24, TRAJ26, TRAJ27, TRAJ28, TRAJ29, TRAJ30,TRAJ31, TRAJ32, TRAJ33, TRAJ34, TRAJ35, TRAJ36, TRAJ37, TRAJ38, TRAJ39, TRAJ40, TRAJ41, TRAJ42,TRAJ43, TRAJ44, TRAJ45, TRAJ46, TRAJ47, TRAJ48, TRAJ49, TRAJ50, TRAJ52, TRAJ53, TRAJ54, TRAJ56,TRAJ57.

10. A genetically modified rodent or rodent cell according to any preceding claim, comprising a TCRP variable gene locus comprising one or more, optionally all, of the following human vp gene segments:TRBV2, TRBV3-1, TRBV4-1, TRBV4-2, TRBV5-1, TRBV5-4, TRBV5-5, TRBV5-6, TRBV6-1, TRBV6-2, TRBV6-4, TRBV6-5, TRBV6-6, TRBV6-8, TRBV7-2, TRBV7-3, TRBV7-4, TRBV7-6, TRBV7-7, TRBV7-9, TRBV9, TRBV10- 1, TRBV10-2, TRBV10-3, TRBV11-1, TRBV11-2, TRBV11-3, TRBV12-3, TRBV12-4, TRBV12-5, TRBV13, TRBV14, TRBV15, TRBV16, TRBV18, TRBV19, TRBV20-1, TRBV24-1, TRBV25-1, TRBV27, TRBV28, TRBV29- 1, TRBV30 and / or comprising one or more, optionally all, of the following human jp gene segments:TRBJ1-1, TRBJ1-2, TRBJ1-3, TRBJ1-4, TRBJ1-5, TRBJ1-6, TRBJ2-1, TRBJ2-2, TRBJ2-3, TRBJ2-4, TRBJ2-5, TRBJ2-6, TRBJ2-7.

11. A genetically modified rodent or rodent cell according to any preceding claim, wherein all of CD3e, CD3y, CD36 and CD3^ are endogenous wild type.

12. A method for making a genetically modified rodent that expresses a human T cell receptor comprising:(i) inserting into an endogenous rodent TCRa variable gene locus an unrearranged human TCRa variable gene locus comprising at least one human Va segment and at least one human Ja segment operably linked to a human TCRa constant region;(ii) inserting into an endogenous rodent TCRP variable gene locus an unrearranged human TCRP variable gene locus comprising at least one human vp segment, at least one human DP segment, and at least one human jp segment operably linked to a human TCRP constant region;(iii) inserting into an endogenous rodent CD8a gene locus a nucleic acid sequence encoding a human CD8a polypeptide;(iv) inserting into an endogenous rodent CD8P gene locus a nucleic acid sequence encoding a human CD8P polypeptide;(v) inserting into an endogenous MHC class I locus a nucleic acid sequence encoding a human MHC class I polypeptide; and(vi) inserting into an endogenous rodent P2 microglobulin gene locus a nucleic acid sequence encoding a human P2 microglobulin, wherein the host loci at the site of each insertion are inactivated or deleted, such that the rodent does not express native rodent TCR, MHC class I or CD8, and optionally also not rodent P2 microglobulin.

13. A method for making a fertile rodent or rodent cell, the method comprising the step of inverting the rodent TCRP in the rodent genome from at least rodent vpi to Dpi, optionally the method further comprising the insertion into a rodent genome of nucleic acid, such as DNA, encoding all or a part of a TCRP from another species which is different from the rodent, such as inserting human DNA encoding all or a part of TCRp.

14. A method for making a genetically modified fertile rodent that expresses a human T cell receptor, the method comprising deletion of the rodent TCR p from at least rodent vpi to Dpi, including a deletion of serine proteases genes Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2, wherein one or more or all of the deleted serine proteases genes Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2 are re-inserted into the rodent genome.

15. A method for making a fertile rodent comprising a genetic knock out of the endogenous T cell receptor beta chain polypeptide, the method comprising deletion of the rodent TCRP from at least rodent vpi to Dpi, including a deletion of serine proteases genes Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2, wherein one or more or all of the deleted serine proteases genes Prss58, Prss59, Prss3b, Try 4, Try5, Try 10, Prss3, Prssl, and Prss2 are re-inserted into the rodent genome.

16. A method for making a fertile rodent, the method comprising, in either order:(i) deletion of the rodent TCRP genomic DNA from at least rodent vpi to Dpi, including a deletion of one or more or all of the serine proteases genes Prss58, Prss59, Prss3b,Try 4, Try5, Try 10, Prss3, Prssl, and Prss2, and(ii) insertion of one or more or all of the serine proteases genes Prss58, Prss59, Prss3b,Try 4, Try5, Try 10, Prss3, Prssl, and Prss2 into the rodent genome.

17. A method according to any of claims 13 to 16, wherein the deletion is homozygous.

18. A rodent or rodent cell, or method for making a rodent or rodent cell according to any one of claims 1-17, wherein the rodent genome comprises DNA encoding a fully human TCRP chain and comprising human V, D J and C gene segments, wherein the rodent or rodent cell is capable of expression of said fully human TCRP chain, and wherein the rodent genome comprises a human intergenic and / or a human intronic DNA sequence located between the human DI and human C2 gene segments, preferably being a human intergenic and / or intronic sequence that naturally occurs together with the human D and J gene segments, optionally wherein all of the human DNA sequence between the human DI and human C2 gene segments is genomic human DNA.

19. A rodent or rodent cell having a genome comprising DNA encoding a fully human TCR beta chain and comprising human V, D, J and C gene segments, wherein the rodent or rodent cell is capable of expression of said fully human TCR beta chain, and wherein the rodent genome comprises a human intergenic and / or a human intronic DNA sequence located between the human DI and human C2 gene segments, preferably being a human intergenic and / or intronic sequence that naturally occurs together with the human D and J gene segments, optionally wherein all of the human DNA sequence between the human DI and human C2 gene segments is genomic human DNA.

20. A method for making a human T cell receptor in a rodent, the method comprising inserting DNA encoding a fully human TCR beta chain and comprising human V, D, J and C gene segments into the rodent genome, wherein the rodent is capable of expression of said fully human TCR beta chain, wherein the insertion comprises a human intergenic and / or a human intronic DNA sequence located between the human DI and human C2 gene segments, preferably being a human intergenic and / or intronic sequence that naturally occurs together with the human D and J gene segments, optionally wherein all of the human DNA sequence between the human DI and human C2 gene segments is genomic human DNA.

21. A method of producing a human T cell receptor to an antigen of interest comprising:(i) optionally, making a rodent according to any preceding claim,(ii) immunizing a rodent of any of the preceding claims with the antigen of interest;(iii) allowing the rodent to mount an immune response;(iv) determining a nucleic acid sequence of the human TCR variable regions expressed by a T cell from the rodent reactive to the antigen of interest, optionally including a step of isolating the T cell and(v) expressing the human T cell receptor, or human T cell receptor variable region in a cell; optionally further formulating the expressed human T cell receptor, or human T cell receptor variable region with a pharmaceutically acceptable excipient; or(vi) inserting the nucleic acid encoding the human T cell receptor, or human T cell receptor variable region, into a cell, ex vivo or in vitro, such as into a human or animal cell, optionally wherein the cell containing the inserted nucleic acid is formulated for delivery to a human or animal, respectively; or(vii) formulating a nucleic acid (e.g. RNA or DNA) encoding the human T cell receptor, or human T cell receptor variable region, with a suitably delivery vehicle, such as a lipid or liposome, for delivery to a patient in need thereof in vivo.

22. A method of treating an individual in need thereof, the method comprising(i) delivery of a soluble TCR molecule comprising the human T cell receptor variable region of step(v) of claim 21, and optionally comprising a human T cell receptor constant region, to the individual,(ii) delivery of a cell according to step (vi) of claim 21 into the patient in need thereof,(iii) delivery of a nucleic acid (e.g. RNA e.g. mRNA or DNA) encoding the human T cell receptor, or human T cell receptor variable region formulated as in step (vii) of claim 21 into a patient in need thereof.

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